nickel utilization by microorganismst · the most intensively examined urease is the plant enzyme...

MICROBIOLOGICAL REVIEWS, Mar. 1987, p. 22-42 Vol. 51, No. 10146-0749/87/010022-21$02.00/0Copyright © 1987, American Society for Microbiology

Nickel Utilization by MicroorganismstROBERT P. HAUSINGER

Department of Microbiology and Public Health and Department ofBiochemistry, Michigan State University,East Lansing, Michigan 48824

INTRODUCTION............................................................... 22NICKEL-CONTAINING ENZYMES ............................................................... 22

Nickel-Containing Ureases ............................................................... 22Bacterial ureases ............................................................... 23Ureases from other ureolytic microorganisms ............................................................... 24

Nickel-Containing Hydrogenases ............................................................... 24Methanogenic bacteria ............................................................... 24Aerobic hydrogen-oxidizing bacteria ............................................................... 27Sulfate-reducing bacteria ............................................................... 28Phototrophic bacteria ............................................................... 29Aerobic nitrogen-fixing bacteria ............................................................... 30Other microorganisms ............................................................... 30

Nickel-Containing Methylcoenzyme M Reductases from Methanogenic Bacteria ................................30Characterization ofF430.................................30Methyl coenzyme M reductase-bound F430.................................. .............................32

Nickel-Containing CO Dehydrogenases...........3.................................................32Acetogenic bacteria ...............................................................33Methanogenic bacteria..........................................o.........34Other bacteria ...............................................................34

OTHER MICROBIAL ROLES FOR NICKEL................................35MECHANISMS OF NICKELUPTAKE.35SUMMARY.................... . .. ........ ................................36ACKNOWLEDGMENTS.................... .............................36.....36LITERATURE CITED................................ ....... ..... o o ....36

INTRODUCTION

Nickel, atomic no. 28, constitutes 8.5% of the earth's coreand about 0.008% of the earth's crust (139). Average world-wide nickel concentrations have been estimated to be 1.0,g/kg for lakes and rivers, 0.6 ,ug/kg for oceans, and 16 ,ug/gfor soil (147). The three predominant nickel isotopes are 58Ni(68.3%), "ONi (26.1%), and 62Ni (3.6%), all having a nuclearspin of zero. In contrast, 61Ni (1.1% abundance) has anuclear spin of 3/2 with a nuclear magnetic moment of-0.749 Bohr magneton (BM). Another experimentally usefulnickel isotope is the ,-emitting 63Ni, with a decay energy of0.067 MeV and a half-life of 92 years. Nickel exists inoxidation states from 0 to +4; however, 0, + 1, and +2 arethe most stable states. Electron paramagnetic resonance(EPR) signals can be observed for the + 1 (d9) and +3 (d7)states of nickel. Hydrogen and carbon monoxide are reactivewith nickel, and this metal is widely used for hydrogenation,carbonylation, and other chemical reactions.

Until recently, nickel has been considered to be biologi-cally significant only because of its toxic effects. In 1965,however, a requirement for nickel was demonstrated byBartha and Ordal during studies with chemoautotrophichydrogen-oxidizing bacteria (24). Subsequent to this discov-ery, nickel ion was shown to be an essential micronutrientfor many microorganisms in which it is incorporated into at

t Michigan State University Agricultural Experiment Station ar-ticle no. 12020.

least four microbial enzymes. These enzymes participate inthe important metabolic reactions of ureolysis, hydrogenmetabolism, methane biogenesis, and acetogenesis. In 1980,a brief review of biological nickel utilization was published(208), and in the same year the microbial transport andmetabolism of nickel was summarized (103). Since that time,the number of microorganisms known to utilize nickel hasgreatly expanded; yet, the physiological roles for this metalion have only begun to be understood. Here an attempt ismade to provide an up-to-date, critical review of studies thathave explored the biological functions of nickel and themechanisms of nickel transport. Topics not discussed herebut reviewed elsewhere include general environmental as-pects of nickel (147), the toxicological effects of nickel onmicroorganisms (20), and the nickel requirements of plants(219) and animals (143). In addition, a forthcoming bookedited by J. Lancaster (Bioinorganic Chemistry ofNickel, inpress) focuses on the biochemistry of nickel and on thespectroscopic tools used to characterize nickel metallocent-ers.

NICKEL-CONTAINING ENZYMESEach of the known nickel-containing enzymes found in

microorganisms is discussed below.

Nickel-Containing Ureases

Urease is found in many plants, algae, fungi, and bacteria,where it degrades urea to ammonia and carbonic acid

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NICKEL UTILIZATION BY MICROORGANISMS 23

TABLE 1. Nickel-containing microbial ureases

Microorganism foEr idencela Subunit Mr contentb Reference(s)

BacteriaArthrobacter oxydans p NRC 0.3/242,000 179Brevibacterium ammoniagenes p 67,000 0.8/67,000 138Bacillus pasteurii p 65,000 1.0/65,000 41Selenomonas ruminantium p 70,000 2.1/70,000 88aKlebsiella aerogenes p, r 72,000, 11,000, 9,000 2.0/72,000 Todd and Hausinger, unpublished data; 179Sporosarcina ureae r NR NR 179Anabaena cylindrica r NR NR 125aOther bacterial ureases i NR 175

FungiAspergillus nidulans r 40,000 NR 44, 124Penicillium sp. r NR NR 162Filobasidiella neoformans var. gatti r NR NR Booth and Vishniac; unpublished dataAspergillus tamarii i NR NR 229Rhodototorula pilimanae i NR NR 5

AlgaePhaeodactylum tricornutum r NR NR 170Tetraselmis subcordiformis r NR NR 170Cyclotella cryptica r NR NR 149

LichenEvernia prunastri r NR NR 155a The evidence for nickel in these enzymes includes the following: p, analysis of purified enzyme; r, nickel requirement for urease activity; i, inhibition by

hydroxamic acids.b Gram-atoms of nickel per M, of subunit or enzyme.c NR, Not reported.

according to equation 1 (14, 171)

011

H2N-C-NH2 + 2H20 -+ 2NH3 + H2CO3 (1)

The most intensively examined urease is the plant enzymefrom jack bean (Canavalia ensiformi) (14, 30). This ureasehas the distinction of being the first enzyme to be crystal-lized, reported by Sumner in 1926 (198). More germane tothis discussion, jack bean urease was also the first enzymedemonstrated to contain nickel (60). The discovery of nickelin jack bean urease originally arose out of critical analysis ofthe enzyme's ultraviolet-visible spectrum which exhibited asmall, but distinct, long-wavelength absorbance (60). Thespectrum has since been assigned to a nickel(II) ion inoctahedral geometry within the enzyme (28). X-ray absorp-tion fine-structure (EXAFS) spectral analysis of urease isconsistent with nickel ligation by oxygen or nitrogen atoms(this technique does not distinguish Ni-O from Ni-N inter-actions) at a distance of 0.204 to 0.294 nm (6). Carefulquantitation by Zerner and colleagues (60) has establishedthe presence of 2 g-atoms of nickel per subunit of hexamericprotein (native Mr = 590,000). An elegant model has beenproposed for the enzyme mechanism wherein one nickel ionpolarizes the urea carbonyl to facilitate nucleophilic attackby an activated hydroxide anion associated with the secondnickel center (14, 30). Nevertheless, proof of the mechanismis lacking, and the detailed nickel environment remainsobscure.

Microbial ureases, important in human health, rumenecology, and soil nitrogen management, are less well char-acterized than the jack bean enzyme. Ureolytic infectionshave been shown to induce urinary deposits (kidney stones)(81, 175), and other dysfunctions, including pyelonephritis,uremic colitis, and hepatic shock resulting from pH elevation

and high ammonia concentration (175). In ruminants, micro-bial ureases participate in nitrogen recycling, in which ureais degraded to provide ammonia for the rumen microbiota(40, 152). In soil, urease activity is essential for releasingammonia from urea-based fertilizers used for plant growth;however, rapid urea degradation by urease can lead to theloss of volatile ammonia and to high-pH-induced crop dam-age (33). Many of the ureolytic microorganisms important ininfection, in the rumen, and in soil have been identified (43,175, 199, 214, 225), and several of their ureases have beenpurified. The known nickel-containing microbial ureases aresummarized in Table 1; the evidence for nickel participationin these enzymes is discussed below.

Bacterial ureases. One of the first indications that bacterialureases contain nickel came from dietary studies in lambs(190, 191); i.e., microbial urease activity in the lamb rumenrequires the presence of this metal ion. More recently,several bacterial ureases were purified and established asnickel-containing enzymes. Using the radioisotope 63Ni,Schneider and Kaltwasser demonstrated that Arthrobacteroxydans urease contains nickel (179). Only 0.3 g-atom ofnickel per enzyme molecule (Mr = 242,000) was found bythis technique, suggesting either contamination of the me-dium with unlabeled nickel or a loss of enzyme-bound nickelduring urease purification. Ethylenediaminetetraacetic acidwas ineffective in removing enzyme-bound nickel; however,nickel was released from the protein and the urease activitywas irreversibly lost at low pH (179). Nakano et al. (138) andChristians and Kaltwasser (41) detected 1 g-atom of nickelper subunit in ureases isolated from Brevibacterium am-moniagenes and Bacillus pasteurii (subunit Mr = 67,000 and65,000, respectively). In contrast, Selenomonas ruminan-tium urease contains 2 g-atoms of nickel per subunit (Mr =70,000) (88a), much like the jack bean enzyme. Furthermore,Klebsiella aerogenes urease appears to possess three protein

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24 HAUSINGER

components (Mr = 72,000, 11,000, and 9,000) and has 2g-atoms of nickel per largest subunit (M. J. Todd and R. P.Hausinger, manuscript in preparation).Other bacterial ureases have been purified, but their nickel

content has not been reported. These preparations includeureases from the enteric organisms Proteus mirabilis (J. A.Anderson, F. Kopko, A. J. Siedler, and E. G. Nohle, Fed.Proc. 28:764, 1969), Morganella morganii (formerly Proteusmorganii) (222), and Providencia rettgeri (formerly Proteusrettgeri) (126), from the gram-positive bacterium Staphylo-coccus saprophyticus (78), and from the blue-green bacte-rium Spirulina maxima (39). Jack bean urease is inhibited byhydroxamic acids and the hydroxamate moiety was shownto bind the metal ion (30). Similarly, several of the aboveenzymes and other bacterial ureases are inhibited byhydroxamic acids (175). Thus, the inhibition results areconsistent with the presence of nickel in a wide range ofbacterial ureases. Further evidence suggesting the presenceof nickel in ureases from K. aerogenes and Sporosarcinaureae was obtained by growth studies in the presence ofethylenediaminetetraacetic acid (179). Under these condi-tions the cells are deficient in urease activity, but activity isrestored by addition of nickel ions to the medium. Thecyanobacterium Anabaena cylindrica also requires nickel inthe medium for urease activity (125a); however, ureaseapoenzyme is generated in nickel-deficient cells. The cyano-bacterial apo-urease can be reconstituted with nickel inwhole cells, but not in cell-free extracts. Neither the enzymenor the apo-enzyme has been purified from this cyano-bacterium.From the evidence summarized above, bacterial ureases

universally appear to contain nickel, but it remains unclearwhy the nickel content varies among the different enzymes.One possibility is that apo-enzyme may copurify with theactive protein in some preparations. Alternatively, only onenickel may be essential for enzyme activity and a second ionis bound noncatalytically in some proteins. Interestingly,nickel was found to inhibit partially purified ureases purifiedfrom K. aerogenes (formerly Aerobacter aerogenes) (104),Staphylococcus saprophyticus (78), and a ruminal mixedmicrobial population (127). This inhibition may be due tometal ion-catalyzed thiol oxidation; no evidence has accruedfor metal ion incorporation during inhibition. No studieshave been reported which probe the active-site metal ioncenter of any bacterial urease. As a result, the environmentaround the nickel center and the role of nickel in enzymeactivity are not known.

Ureases from other ureolytic microorganisms. Fungi maypossess high levels of urease; e.g., in Aspergillus tamarii theenzyme can account for as much as 8.5% of the total solubleprotein (229). The high urease content in Aspergillus tamariiis not required for enzyme activity, but rather, the protein isthought to serve as a reserve nitrogen source for the micro-organism. The only two fungal ureases that have beenpurified are the enzymes from Aspergillus tamarii (229) andAspergillus nidulans (44). The nickel content was not re-ported for either of these proteins.MacKay and Pateman (125) identified four genes in

Aspergillus nidulans (ureA, ureB, ureC, and ureD) that arerequired for urease activity. Their results suggested thatureA is involved with urea transport and ureB encodes theurease structural protein. The functions of ureC and ureDare not established. However, since 0.1 mM nickel cansupport growth of a ureD mutant, the authors suggested(124) that ureD may function to synthesize a nickel cofactoror to incorporate nickel or a nickel cofactor into the struc-

tural enzyme. No direct evidence for a nickel cofactor hasbeen reported for any urease. An alternative role for theureD gene may be specific nickel transport. Similar to theAspergillus nidulans system, urease mutations of Neuros-pora crassa were assigned to four loci, occurring in twoclosely linked pairs (25). In contrast to the complex geneticsjust described, only two loci were found to be involved inurease activity of Ustilago violaceae (21). One of the U.violaceae genes encodes the structural protein, the secondmay involve a urea permease, and no evidence was pre-sented for a gene involving nickel utilization.

Several nongenetic studies have suggested an associationbetween nickel and fungal ureases. For example, theAspergillus tamarii (229) and Rhodototorula pilimanae (5)enzymes are inhibited by primary hydroxamic acids. Theurease activity of Filobasidiella neoformans var. gatti wasshown to be inhibited by ethylenediaminetetraacetic acidand reactivated by nickel (J. L. Booth and H. S. Vishniac,Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, K206, p.227). Further, nickel is required for urea-supported growthby a fungus, Penicillium sp. (162), and a lichen, Everniaprunastri (155). It is not known whether the fungus or theassociated phototroph is the source of lichen urease. Incontrast to these reports, Phycomyces blakesleeanus ureaseexhibits a dependence on zinc ion (90). The significance ofthis result is unclear, and the zinc-dependent enzyme has notbeen purified or characterized.

Algae that belong within the Chlorophyceae and manytypes of yeasts hydrolyze urea by a pathway that involves anadenosine triphosphate (ATP) and biotin-dependent ATP-urea amidolyase (173, 174). This enzyme apparently does notrequire nickel. In contrast to these green algae, Cyclotellacryptica (149), Phaeodactylum tricornutum (170), andTetraselmis subcordiformis (170) have been shown to re-quire nickel for growth on urea. As is the case for allnickel-containing microbial ureases, several concerns re-main to be elucidated; e.g., the number of nickel atoms peractive site, the postulated presence of a nickel cofactor, andthe detailed environment and role of nickel.

Nickel-Containing Hydrogenases

Hydrogenases catalyze the following deceptively simpleoxidation-reduction reaction (equation 2)

H2 ; 2H+ + 2e- (2)These enzymes are often classified according to their in vivorole catalyzing either hydrogen-consuming or hydrogen-evolving reactions. The hydrogenase literature was thor-oughly reviewed in 1981 (3), and the physiological roles,some of the properties, and the postulated enzyme mecha-nisms were discussed. Since then, many of the uptakehydrogenases were found to possess nickel (36, 134). Theevidence for nickel participation in microbial hydrogenasecatalysis is discussed below, and a summary of the knownnickel-containing hydrogenases is provided in Table 2.Methanogenic bacteria. Hydrogenases play a central role

in many methanogenic bacteria in which hydrogen oxidationis used to drive reduction of carbon dioxide and generateenergy for the cell (48). In all cases examined, methanogenhydrogenases contain nickel. Interest in the role of nickel inthese hydrogenases began with the work of Lancaster (115)with Methanobacterium bryantii cell extracts. In oxidizedmembrane samples, EPR spectroscopy revealed a novelparamagnetic signal with g values of 2.30, 2.23, and 2.02

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TABLE 2. Nickel-containing microbial hydrogenases

Microorganism Forma Ev-b Subunit Mr Metal and cofactor contentc Reference(s)Methanogenic bacteriaMethanobacterium thermoautotrophicum Soluble, B

(Marburg)Methanobacterium thermoautotrophicum Soluble, A(AH)

Methanobacterium formicicum

Methanosarcina barkeriMethanococcus vannielii

Methanobacterium bryantii

Soluble, BSoluble, A

Soluble, B

Soluble, ASoluble, A

Membrane bound

p NRd

p 47,000, 31,000,26,000

s 52,000, 40,000p 42,600, 34,000,

23,500p 48,000, 38,000

p 60,000p NR

s NR

0.8 Ni/60,000

0.64 Ni, 13.4 Fe, 0.83FAD/115,000

NR3 Ni, 20 Fe/170,000

0.49 Ni, 9.4 Fe, 0.8 Zn, 1.6Cun0,000

0.7 Ni, 9 Fe, 1 FMN/60,0002 Ni, 3.8 Se/340,000NR

80

110

110101, 141

1, 101

68Yamazaki, un-

published data116

Hydrogen-oxidizing bacteriaAlcaligenes eutrophus Soluble

Alcaligenes eutrophus Membrane boundAlcaligenes latus Membrane boundNocardia opaca Soluble

Xanthobacter autotrophicus NRPseudomonas flava NRArthrobacter sp. strain 11X NR

Sulfate-reducing bacteriaDesulfovibrio gigas PeriplasmicDesulfovibrio desulfuricans (ATCC 27774) SolubleDesulfovibrio desulfuricans (Norway) SolubleDesulfovibrio desulfuricans (Norway) Membrane boundDesulfovibrio multispirans CytoplasmicDesulfovibrio baculatus PeriplasmicDesulfovibrio salexigens PeriplasmicDesulfovibrio africanus SolubleDesulfovibrio vulgaris Membrane bound

p 63,000, 56,000,30,000, 26,000

p 61,000, 30,000p 67,000, 34,000p 64,000, 56,000,

31,000, 27,000r NRr NRr NR

p 62,000, 26,000p NRp 56,400, 28,600s 60,000, 27,000p 58,000, 24,500p 100,000p 62,000, 36,000p 65,000, 27,000p 86,000, 45,000e

2 Ni, 16 Fe, 1 FMN/205,000 74, 91

0.65 Ni, 8 Fe/98,000 74, 1810.54 Ni, 1.7 Fe/101,000 1613.8 Ni, 13.6 Fe, 1 FMN/178,000 180

NR 137, 201NR 201NR 201

1 Ni, 12 Fe/89,500 37, 1180.6 Ni, 10.9 Fe/75,500 1111 Ni, 8 Fe, 1 Se/85,000 172Ni, 6 Fe/58,000 114, 1720.9 Ni, 11 Fe/82,500 451 Ni, 1 Se/100,000 2051 Ni, 12-15 Fe, 1 Se/98,000 2050.9 Ni, 12 Fe/92,000 1440.3 Ni, 4 Fe, 0.3 Se/130,000 120

Phototrophic bacteriaChromatium vinosumRhodobacter capsulataThiocapsa roseopersicinaAnabaena cylindricaAnabaena variabilisAnabaena sp. strains CA and 1FAnabaena sp. strain 7119Anacystis nidulansMastiglocladus laminosus

Aerobic nitrogen-fixing bacteriaBradyrhizobium japonicumAzotobacter vinelandiiAzotobacter chroococcomAzospirillum brasilenseAzospirillum lipoferumDerxia gummosa

Other bacteriaWolinella succinogenesEscherichia coli

SolubleMembrane boundMembrane boundNRNRNRNRNRNR

Membrane boundMembrane boundNRNRNRNR

Membrane boundMembrane bound

p 62,000p 65,000p 47,000, 25,500r NRr NRr NRr NRr NRr NR

p 64,000, 35,000p 67,000, 31,000r NRr NRr NRr NR

1 Ni, 4 Fe/62,0000.2 Ni, 3.6 Fe/65,0001 Ni, 4 Fe/68,000NRNRNRNRNRNR

0.6 Ni, 6.5 Fe/104,0000.7 Ni, 6.6 Fe/98,000NRNRNRNR

p 60,000, 30,000 1 Ni, 11-20 Fe/100,000p 64,000, 35,000e 0.6 Ni, 12 Fe/200,000

74279, 2324713227150150153

17, 86, 195187151154154154

21022, 177

a For the methanogenic bacteria, A represents an F420-reducing hydrogenase and B represents a non-F420-reducing hydrogenase.b The evidence for nickel in these enzymes includes the following: p, analysis of purified enzyme; r, nickel required for hydrogenase activity; s, spectroscopic

identification.c Gram-atoms of metal per Mr of minimal form of the enzyme. FAD, Flavin adenine dinucleotide.d NR, Not reported.eAt least two other forms of nickel-containing hydrogenase have been reported for this microorganism.

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TABLE 3. Selected nickel EPR spectroscopic signals from nickel-containing enzymes

SignalEnzyme Microorganism State of enzyme g valuea intensity Reference(s)

(%)b

Hydrogenase Methanobacterium thermoautotro- 1. Aerobic 2.309, 2.237, 2.017 50 10, 110phicum 2. Reduced with hydrogen, then 2.196, 2.140 NR 110

incubated under argonAlicaligenes eutrophus (membrane- 1. Aerobic 2.30, 2.17, 2.01 15 181bound enzyme) 2. Reduced several hours under 2.25, 2.11, 2.05 NR 35

hydrogenDesulfovibrio gigas 1. Native, aerobic 2.31, 2.23, 2.02 40-50 37, 118

2. Partial reduction by hydrogen 2.19, 2.16, 2.02 10-25 118, 1333. Reduced, followed by dye 2.33, 2.16, 2.02 NR 38, 70

oxidationChromatium vinosum 1. Ascorbate/phenazine methosulfate (I) 2.337, 2.164, NR 11, 213

treated or partial reduction by 2.011cdithionite (II) 2.321, 2.241,

2.0112. Dithionite reduced 2.20, 2.15, 2.01 NR 2133. Dithionite reduced illuminated (I) 2.30, 2.13, 2.04c NR 213

(II) 2.28, 2.13, 2.04Methyl coenzyme Methanobacterium thermoautotro- 1. Aerobic 2.233, 2.168, 2.154 3-16 8

M-reductase phicumCO dehydrogenase Clostridium thermoaceticum 1. Anaerobic, dithionite-free 2.21, 2.11, 2.02 10 166

2. CO treated (I) 2.074, 2.074, 20-50d 165, 166,2.028c 168, 169

(II) 2.062, 2.047,2.028

3. CO treated plus coenzyme A 2.074, 2.074, 2.028 50d 168, 169a The g values refer only to EPR spectroscopic signals involving nickel. Other spectral signals are often present which arise from iron-sulfur centers, flavins,

or other enzyme components. Detailed EPR spectroscopic conditions are provided in the references.bThe integrated intensity of the nickel signals are reported as percentage of nickel concentration in the samples. NR, Not reported.c Two types of signals are observed and vary in relative intensity between various preparations.d The signal is suggested to arise from a spin-coupled complex containing nickel, iron, and carbon (169).

attributed to octahedral coordination of nickel(III). Thisassignment was corroborated by studies in which Lancastergrew the microorganism in the presence of 6tNi possessing anuclear spin of 3/2. The EPR spectrum of oxidized cellextracts from the 6tNi-grown cells exhibited clearly definedhyperfine structure, as expected for a signal due to nickel(116). Similar EPR spectroscopic signals were also observedby Lancaster in cell extracts from Methanobacteriumthermoautotrophicum and Methanosarcina barkeri (117),and more recently, Albracht et al. (8) demonstrated that thisnovel nickel(III) signal could be observed in whole cells ofMethanobacterium thermoautotrophicum strain Marburg.No nickel signals, however, were observed in cell extractsfrom Methanobrevibacter ruminantium, Methanococcusvannielii, or Methanospirillum hungatei (117).The nickel(III) paramagnet observed in whole cells or in

cell extracts was finally shown to be associated with cellularhydrogenases in studies on the purified enzymes. The firstmethanogen hydrogenase to be purified to near homogeneitywas from Methanobacterium thermoautotrophicumMarburg (80). In this study, Graf and Thauer (80) grew theorganism with 63Ni and found that the radioactivitycopurified with both forms of benzyl viologen-reducinghydrogenase. The major enzyme fraction was found topossess 0.8 g-atom of nickel per enzyme molecule (Mr =60,000) (80) and to exhibit an EPR spectroscopic signalarising from nickel(III) (10). The nickel signal disappearedwhen the oxidized enzyme was placed under hydrogen gas;hence, the hydrogenase nickel center is redox active (10).The natural electron acceptor of this benzyl viologen-reducing, nickel-containing hydrogenase has not been iden-tified, and it is unclear whether this hydrogenase is involvedin methanogenesis.

Although Methanobacterium thermoautotrophicumstrains AH and Marburg share a common species assign-ment, these cultures are not closely related based on deox-yribonucleic acid hybridization (32). In contrast to the singlehydrogenase studied in the Marburg strain, two solublehydrogenases have been isolated from the AH strain, andboth enzymes possess redox-active nickel (110). Only one ofthese enzymes is able to reduce coenzyme F420, a two-electron carrier found in methanogens. The F420-reducinghydrogenase is composed of three subunits with Mr =47,000, 31,000, and 26,000, and the best preparations contain0.64 g-atom of nickel, 13.4 g-atom of iron, and 0.83 mol offlavin adenine dinucleotide based on a minimum Mr of115,000. Localization of nickel to the subunit of Mr 47,000was suggested by mild detergent dissociation of native63Ni-labeled F420-reducing hydrogenase (C. Walsh, personalcommunication). The second hydrogenase does not reduceF420, but rather was assayed by methyl viologen reduction(110). The second enzyme contains nickel and iron but noflavin, and the protein is composed of two subunits with Mr= 52,000 and 40,000. The oxidized forms of bothMethanobacterium thermoautotrophicum AH hydrogenasesgive rise to a nickel(III) EPR spectrum (Table 3; Fig. 1). Thenickel signals for both enzymes disappear during incubationwith hydrogen. On the other hand, with the F420-reducingenzyme, a new nickel EPR spectral signal (g values = 2.20,2.14, and 2.0) is formed when hydrogen is replaced by argon.This state of the enzyme may be similar to the anaerobicoxidized form of hydrogenase within the cell. This experi-ment provides compelling evidence that a nickel(III) statemay occur during a normal catalytic cycle.The nickel-containing active sites of the two Methano-

bacterium thermoautotrophicum AH hydrogenases have

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g-value

FIG. 1. Nickel EPR spectroscopic signal of a methanogen hy-drogenase. The upper spectrum was recorded at 150 K for theaerobic F420-reducing hydrogenase isolated from Methanobacteriumthermoautotrophicum AH and illustrates the g values typical formany nickel hydrogenases (g = 2.31, 2.24, 2.02). The lower spec-trum was recorded for the same enzyme isolated from a culturewhich was grown in media enriched in 6"Ni, a nickel isotope withnuclear spin I = 3/2. The clearly defined hyperfine structure of thisspectrum provides unambiguous evidence for the presence of para-magnetic nickel. (Adapted from reference 110).

also been characterized by using other approaches. EXAFSspectroscopy was used to demonstrate that at least threesulfur atoms serve as ligands to nickel at a distance of 0.225

0.004 nm (119). No shorter bond distances were observed;thus, strong interactions between nickel and oxygen ornitrogen were eliminated. The nickel-sulfur charge transferbands of the nickel(III) center in oxidized hydrogenases gaverise to a low-temperature magnetic circular dichroism spec-trum (102). A nitrogen atom does weakly interact with thenickel center in the F420-reducing hydrogenase, but not themethyl viologen-reactive enzyme, as deduced by electronspin echo spectroscopy (203). Figure 2 presents a model ofthe nickel center in these ansd other nickel-containinghydrogenases, based on EPR and EXAFS studies. Althoughthe Methanobacterium thermoautotrophicum AH F420-reducing enzyme is the best-characterized methanogen hy-drogenase, many questions clearly must be answered aboutthe role and environment of the nickel-active site in theprotein.

Nickel was shown by Nelson et al. (141) to be a compo-nent of the Methanobacterium formicicumn F420-reducinghydrogenase. More recently, Adams et al. (1) examined theredox properties of an F420-nonreactive hydrogenase fromMethanobacteriumformicicum. The best preparations of thedimeric protein (native Mr = 70,000) contain 0.49 g-atom ofnickel, 9.4 g-atoms of iron, 0.8 g-atom of zinc, and 1.6g-atoms of copper per enzyme molecule. A nickel(III) EPRspectrumn was observed with the oxidized hydrogenase, andthe signal disappeared during incubation under hydrogen.Reoxidation of the reduced enzyme led to a new spectralspecies which was assigned as a copper(II) signal. Thissurprising finding of redox-active copper in Methano-bacteriumformicicum hydrogenase led the authors to searchfor copper in other methanogen hydrogenases. Subse-

quently, copper and zinc were found in both hydrogenases ofMethanobacterium thermoautotrophicum (1), yet no copperEPR spectral signals were observed. The roles of copper,zinc,, and nickel in these proteins are not yet established.Other methanogen hydrogenases have been purified (Ta-

ble 2), but they are generally less well characterized.Methanosarcina barkeri possesses an F420-reducing enzymewhich is closely related to the F420-reducing hydrogenasesdescribed above. The best preparations of the M. barkerienzyme contains 0.6 to 0.8 g-atom of nickel, 8 to 10 g-atomsof iron, and about 1 mol of flavin mononucleotide (FMN) perprotein molecule (Mr = 60,000) (68). A nickel(III) EPRspectroscopic signal was observed for the oxidized hydrog-enase, and the signal disappeared from the sample underhydrogen gas. The Methanosarcina barkeri enzyme is theonly hydrogenase that has been purified from an acetoclasticmethanogen, and its in vivo role is unknown. An F420-reducing hydrogenase from Methanococcus vannielii hasbeen shown to contain nickel as well as selenium (S.Yamazaki Fed. Proc. 42:2977, 1983). The role of the metalions in the protein, however, is not known.

Aerobic hydrogen-oxidizing bacteria. The aerobic hydro-gen-oxidizing "Knallgas" bacteria include representativesof Alcaligenes, Pseudomonas, Paracoccus, Aquaspirillum,Xanthobacter, Nocardia, Mycobacterium, and Bacillus spe-cies (3). In nearly all of these bacteria, a membrane-boundhydrogenase is linked to a respiratory chain and is coupledwith ATP generation. In addition, a soluble enzyme ispresent in some species which is nicotinamide adeninedinucleotide (NAD) linked and primarily involved in reduc-tive fixation of CO2 (3).A nickel requirement for chemolithotrophic growth of two

strains of Alcaligenes sp. (formerly Hydrogenomonas) wasdemonstrated over 20 years ago (24). This initial observationwas followed by the studies of Tabillion et al. (201), whodemonstrated nickel-dependent chemolithotrophic growth offive strains of Alcaligenes eutrophus, two strains ofXanthobacter autotrophicus, Pseudomonas flava, andArthrobacter sp. strains llx and 12x. Similarly, Nakamuraet al. (137) demonstrated a nickel growth dependence ofanother strain of X. autotrophicus. In contrast, Paracoccusdenitrificans and Nocardia opaca lb did not require nickelfor growth (201). This latter finding was surprising because

X

X'S NiX-SzI-0

X

..S-X

x

FIG. 2. Structural model for the hydrogenase nickel center. EPRspectroscopic analyses for many hydrogenases suggest a distortedoctahedral geometry at the nickel center. EXAFS spectroscopy hasbeen used for identification of sulfur as the predominant nickelligands. The source of these sulfur ligands (inorganic sulfur, cysteinethiol) and the identity of the axial ligands are unknown.

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purified Nocardia opaca lb hydrogenase was known torequire nickel for stable activity (4).A relationship between hydrogenase activity and nickel

dependence was observed by Friedrich et al. (72), whodemonstrated a significant reduction of Alcaligeneseutrophus hydrogenase activity when the cells were culturedin the absence of nickel. Both membrane-bound and solubleenzyme activities were affected, and the hydrogenase activ-ity was restored by synthesis of new enzyme after theaddition of nickel (72, 75). Radioactive 63Ni was used toshow that nickel taken up by Alcaligenes eutrophus wasincorporated specifically into the two hydrogenases (74).Both of the nickel-containing hydrogenase enzymes havebeen purified and characterized.The soluble, NAD-linked hydrogenase from Alcaligenes

eutrophus H16 is a tetramer consisting of four nonidenticalsubunits with Mr = 63,000, 56,000, 30,000, and 26,000 (182).X-ray fluorescence analysis demonstrated the presence of 2g-atoms of nickel per enzyme molecule (74). In addition, theprotein contains one flavin and multiple iron-sulfur centers.Hornhardt et al. suggested that the nickel is located in thesubunit with Mr = 56,000 (91), based on studies of themutant strain HF14. This mutant is defective in hydrogenaseactivity and appears to produce only the hydrogenasesubunit of Mr 56,000. The HF14 protein was purified on thebasis of antibody cross-reactivity with antibody directedtoward the wild-type subunit and was found to contain 0.2 to1.4 g-atoms of nickel and 2 to 3 g-atoms of iron per subunit.Although the HF14 protein was inactive when assayed withNAD, weak activity was observed with several other elec-tron acceptors. Thus, the actual site of hydrogen oxidationmay be located in this subunit. No EPR spectroscopic signalfor nickel was observed for either the wild-type enzyme orthe mutant protein (91).The membrane-bound hydrogenase of Alcaligenes

eutrophus H16 also has been purified and shown to containnickel. This enzyme is composed of two subunits with Mr =61,000 and 30,000 and possesses 0.6 to 0.7 g-atom of nickeland 7 to 9 g-atoms of iron per hydrogenase molecule (181).Of great interest, the large subunit of the membrane-boundenzyme exhibits significant cross-reactivity with antibodydirected toward the subunit of Mr = 56,000 from the solublehydrogenase (182). Since this subunit of the soluble enzymeis suggested to contain nickel (91), by analogy, the largesubunit of the membrane-bound hydrogenase may also con-tain the nickel-binding site. The air-oxidized enzyme givesrise to a weak nickel(III) EPR spectroscopic signal (Table 3)with g values of 2.30, 2.17, and 2.01 (181). This signal wassuggested to arise from an inactive state of the hydrogenasewhich may be converted to the fully activated state by acycle of dithionite reduction and K3Fe(CN)6 reoxidation inthe presence of mediator dyes. Under the latter conditions,a new signal (g = 2.02) is observed which may arise from theinteraction of an iron-sulfur center with a paramagnet, suchas nickel(III) (181). Enzyme which is reduced by hydrogenfor several hours gives rise to a complex EPR spectrum,including a possible nickel-derived signal at g = 2.25,2.11, and 2.05 (35). Further studies are needed to unravelthe complicated EPR spectral properties of this hydro-genase.

Alcaligenes latus contains a membrane-bound hydroge-nase when growing autotrophically with hydrogen and car-bon dioxide, as well as when growing heterotrophically withdinitrogen as sole nitrogen source. The enzyme is identicalfor both growth conditions and it is similar to the Alcaligeneseutrophus membrane-bound hydrogenase. The purified pro-

tein is composed of two subunits with Mr = 67,000 and34,000 and has a metal content of 0.54 g-atom of nickel and1.7-g-atoms of iron per enzyme molecule (161). The lowmetal content is thought to be due to partially inactivesample. No further characteristics of the nickel metal-locenter have been reported for this hydrogenase.Nocardia opaca lb is atypical of the hydrogen-oxidizing

bacteria by not forming membrane-bound hydrogenase. Thesoluble enzyme, however, has been purified (4, 180, 183) andconsists of four subunits with Mr = 64,000, 56,000, 31,000,and 27,000, similar to the Alcaligenes eutrophus solublehydrogenase (180). Each molecule of Nocardia opaca en-zyme has 4 g-atoms of nickel, 14 g-atoms\of iron, and 1 molof FMN. This is twice the nickel content of the Alcaligeneseutrophus hydrogenase. Nickel was found to play two rolesin the Nocardia opaca enzyme; i.e., 2 tightly-bound nickelatoms function as in the Alcaligenes eutrophus hydrogenaseand 2 dissociable nickel atoms are involved in activating theenzyme by inducing dimer association. This activation canalso be achieved by the presence of a very high salt concen-tration (183). In the absence of nickel or high salt, the activeenzyme dissociates to yield two inactive dimers. This inac-tivation mechanism is not observed in any other hydroge-nase. One dimer contains subunits of Mr = 64,000 and 31,000and possesses three iron-sulfur centers and FMN. A seconddimer contains subunits with Mr = 56,000 and 27,000 andpossesses one iron-sulfur center and 2 g-atoms of nickel.This dimer is unable to reduce NAD, but can reduce severalother electron acceptors. Hence, the site for reaction withhydrogen seemed to be localized in the nickel-containingdimer (180). No EPR spectroscopic signals arising from thisenzyme could be directly assigned to nickel; however, as inthe soluble Alcaligenes eutrophus hydrogenase case, thepartially reduced Nocardia opaca enzyme gives rise to a g =2.01 signal, which may indicate an interaction between aniron-sulfur center and paramagnetic nickel (180).

Sulfate-reducing bacteria. Sulfate-reducing bacteria pos-sess high levels of hydrogenase activity. The bioenergeticsystems of these bacteria are intimately coupled to theircellular hydrogenases, as described by Odom and Peck(148). Desulfovibrio sp. has a unique ability to grow inconsortia by interspecies hydrogen transfer in which thesulfate reducer can either produce or utilize hydrogen.Moreover, cytoplasmic hydrogen production and periplas-mic hydrogen consumption may occur simultaneously in thesame microorganism (148). Such a hydrogen-recyclingmechanism would bestow an attractive energetic advantageon this microorganism. The two hydrogenases required forsuch a scheme have been observed in several species ofsulfate-reducing bacteria.Hydrogenases have been isolated and partially character-

ized from several sulfate-reducing bacteria. A list of thosethat contain nickel is shown in Table 2. The best-characterized nickel-containing hydrogenase of the sulfate-reducing bacteria is the enzyme from Desulfovibrio gigas(36, 134). The properties of the nickel site in this enzyme aresummarized below.The periplasmic hydrogenase from D. gigas possesses two

subunits with Mr = 62,000 and 26,000 and is thought tocontain 1 g-atom of nickel and 12 g-atoms of iron per enzymemolecule (Mr = 89,500) (37, 118). As in the case of theMethanobacterium thermoautotrophicum hydrogenase, fournickel ligands were identified as sulfur atoms at a distance of0.22 nm by using EXAFS spectroscopy (186). Two groupssimultaneously reported an intense, isotropic EPR spectralsignal (g = 2.02) from an iron-sulfur center and a second,

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rhombic signal (Table 3) arising from nickel(III) in the nativeoxidized D. gigas enzyme (37, 118). Proof that nickel wasspectrally involved was demonstrated by hyperfine interac-tion in the EPR spectrum of 61N-enriched hydrogenase (133).The enzyme is relatively inactive as isolated; however,incubation under hydrogen slowly restores activity. Thetime-dependent increase in enzyme reactivity was reflectedin a complex series of EPR spectral changes recorded for thehydrogenase (35, 38, 70, 206, 207). Initially, hydrogen reduc-tion led to a loss of both the nickel(III) signal and the g =2.02 iron-sulfur center signal. Further reduction resulted inthe appearance of an EPR spectral signal attributed tonickel, but with altered g values (Table 3) (133). Finally, thecompletely reduced enzyme exhibited a spectrum due onlyto iron-sulfur centers. Cammack and colleagues (38, 70) haveproposed that these spectral changes represent transforma-tions of nickel from nickel(III) to nickel(II) to nickel(I) to anickel(II)/hydride species. In contrast, Teixeira et al. (207)have suggested that these spectra represent changes fromnickel(III) to an EPR silent, spin-coupled nickel(III)/iron-sulfur center species to a nickel(III)/hydride intermediate toa nickel(II) state. Although the interpretations differ, bothgroups report that the nickel is redox active and appears tointeract with the substrate. The reduced hydrogenase couldbe anaerobically reoxidized by using 2,4-dichlorophe-nolindophenol to yield yet another nickel(III)-containingspecies, which was able to be activated without a lag period(70). This state is converted to the initially describednickel(III) species in the presence of oxygen.The reduction potential for certain changes in D. gigas

hydrogenase have been estimated by several methods. Cam-mack et al. (37) titrated the native enzyme by using sodiumdithionite and observed a pH-independent one-electron re-duction for the g = 2.02 signal at -35 mV (versus H2/H'couple) and a pH-dependent one-electron reduction for thenickel(III) signal at -145 mV (pH 7.2). A pH dependencewas also observed by Fernandez et al. (69) and Lissolo et al.(121) for the reduction potential associated with enzymeactivation; however, these values were much lower at -360and -310 mV (pH 7), respectively. In contrast to thesestudies, Mege and Bourdillon (129) examined the reductionpotential for the anaerobic dye-oxidized nickel(III) state.Activation of the hydrogenase was rapidly achieved at apotential of -210 mV (pH 8.3) and again exhibited a pHdependence with a change of 60 mV per pH unit. The resultsfrom these reduction potential studies, when combined withthe EPR spectral analyses, are beginning to form a coherentpicture of the hydrogenase nickel center. However, thedetailed role for nickel remains to be elucidated.The membrane-bound hydrogenase from D. desulfuricans

(Norway) (114) and the soluble enzymes from D.desulfuricans (ATCC 27774) (111) and D. multispirans (45)are similar to the D. gigas protein in that they exhibitnickel(III) and isotropic g = 2.02 EPR signals in theiroxidized states. In contrast, the periplasmic hydrogenasesfrom D. baculatus (205) and D. salexigens (205) and thesoluble enzymes from D. desulfuricans (Norway) (172) andD. africanus (144) are EPR silent when isolated. These latterproteins resemble the Alcaligenes eutrophus soluble hydrog-enase in their EPR properties and in their ability to berapidly activated (35, 205). The slow to be activated enzymesexhibiting a nickel(III) EPR signal may represent oxidativelydamaged forms which are not typically observed in the cell.Not all hydrogenases in sulfate-reducing microorganisms

contain nickel, and a nickel-free, periplasmic hydrogenasefrom D. vulgaris has been extensively characterized (95).

However, Lissolo et al. (120) recently ascertained the pres-ence of three additional membrane-bound hydrogenases inthis microorganism; each of these intrinsic membrane pro-teins appears to contain nickel (120).

Phototrophic bacteria. Two recent publications summarizethe literature on hydrogenases of photosynthetic microorga-nisms (79, 215). These enzymes have been purified andcharacterized from purple sulfur bacteria, purple nonsulfurbacteria, green bacteria, cyanobacteria, and green algae. Asdescribed below and summarized in Table 2, many of theseuptake hydrogenases contain nickel.The most extensively studied hydrogenase from a

phototrophic bacterium is that from Chromatium vinosum.Various preparations of the enzyme have been described;however, a soluble form (Mr = 62,000) is the best charac-terized. Albracht et al. (7) suggested in 1982 that nickel(III)may be present in the aerobic enzyme where it is magneti-cally coupled to an iron-sulfur cluster. This hypothesis wasstrengthened by a series of EPR spectral studies at 4, 9, and35 GHz, and the two spin systems were estimated to beseparated by no more than 1.2 nm (12). Interaction betweenthe metallocenters could be chemically interrupted, resultingin inactivated enzyme and the observation of a nickel(III)EPR spectrum (Table 3) (11). However, the crucial magneticcoupling between the nickel and iron centers could berestored by incubation under hydrogen with reducing agentsat 50°C (9). Reductive titration of the enzyme by usingdithionite led to a series of spectral changes in which anickel(III) type of signal appeared at -38 mV and disap-peared at -175 mV, and a new signal was observed below-300 mV (213). This latter signal was assigned to a nickel(I)state, perhaps with an axial hydride; however, unambiguousidentification is not yet possible. The putative nickel(I)valence state is light sensitive and the spectroscopicallyobserved photoreaction occurs sixfold more slowly in 2H20than in H20 (213). This result is consistent with the presenceof nickel-hydride coordination in this state and may relate tothe role of nickel in the enzyme. Additional evidence sup-porting a role for nickel in hydrogenase is derived frominhibition studies; i.e., carbon monoxide, a competitiveinhibitor, appears to bind to the same nickel ligand positionas hydrogen (212). Further studies are needed to fullycharacterize the various states of nickel in the Chromatiumvinosum hydrogenase.The hydrogenases of Rhodobacter capsulata (formerly

Rhodopseudomonas capsulata) (42) and Thiocapsaroseopersicina (79, 232) each probably contain 1 g-atom ofnickel per enzyme molecule. Perhaps as a result, thehydrogenases from each of these purple bacteria are stimu-lated by nickel (202, 232). The green bacterium Chlorellaemersonii also has been shown to require nickel for growth;however, a role for nickel in its hydrogenase has not beendescribed (189).

Nickel-dependent growth of Oscillatoria sp. was de-scribed in 1978 (211); however, the specific role of the metalwas not determined. Since these studies, nickel-dependenthydrogenase activities have been shown in othercyanobacteria. These bacteria include Anabaena cylindrica(47), Anabaena variabilis (13), Anabaena sp. strains CA and1F (227), Anabaena sp. strain 7119 (150), Anacystis nidulans(150), and Mastigocladus laminosus (153). However,hydrogenases from these cells have not been purified andcharacterized, with the exception of the partially purifiedAnabaena sp. strain 7119 enzyme (150). Each of thesecyanobacteria fixes nitrogen and their hydrogenases mayhave an energy-conserving role in this process (see a more

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complete description in the following section). However,whether or not hydrogenase activity enhances the nitrogen-fixing capability of these microorganisms depends on thegrowth conditions (46, 153).

Aerobic nitrogen-fixing bacteria. The uptake hydrogenasesfrom aerobic nitrogen-fixing microorganisms recently havebeen reviewed by Arp (18). These enzymes may provide asubstantial benefit to the cells by improving the efficiency ofnitrogen fixation. In addition to catalyzing dinitrogen reduc-tion, nitrogenase reduces protons to hydrogen at the expenseof ATP. Thus, the uptake hydrogenases allow some of thisenergy to be recovered by the cell. In addition to serving asthe reductant in nitrogen fixation, the hydrogen can be usedfor respiration. In turn, the hydrogen respiration may lowerthe oxygen content of the culture and thereby protect thenitrogenase enzymes from oxygen inactivation. Finally, athird postulated benefit from hydrogenase uptake activity isto decrease the cell concentration of hydrogen, which itselfis an inhibitor of nitrogen fixation. Additional evidence thatuptake hydrogenases can enhance the nitrogen-fixing capac-ity of microorganisms is not discussed in this report. Rather,the evidence that these enzymes contain nickel is summa-rized.Bradyrhizobium japonicum (formerly Rhizobium

japonicum) is an extensively studied microorganism becauseit serves as a model for nitrogen-fixing symbiosis whenassociated with soybeans. In the free-living state, nickel isrequired for hydrogen-dependent growth of Bradyrhizobiumjaponicum (109). Hydrogenase was purified from free-living63Ni-grown cells, and radioactivity comigrated with hydrog-enase activity during gel electrophoresis (86, 195). In thesestudies, strictly anaerobic procedures were used duringenzyme preparation. However, a more convenient aerobicisolation procedure is now available that uses reactive red120-agarose affinity chromatography (194). The Brady-rhizobium japonicum hydrogenase purified from soybeanroot nodules is apparently identical to the enzyme in free-living cells (17). Purified uptake hydrogenase is a dimeric,membrane-bound protein composed of subunits with Mr =64,000 and 35,000 and contains a 0.6 g-atom of nickel and 6.5g-atoms of iron per enzyme molecule. This hydrogenase hasbeen shown to be immunologically related to the membrane-bound enzymes from Alcaligenes eutrophus, Alcaligeneslatus, and Azotobacter vinelandii (19). An antigenic relation-ship to the Escherichia coli hydrogenase has also beenreported for the Bradyrhizobium enzyme (87). The twospecies of Alcaligenes were discussed in the hydrogen-oxidizing bacteria section above, and their hydrogenases areknown to contain nickel. Other evidence, discussed below,suggests that the Azotobacter vinelandii and Escherichia colienzymes also contain nickel.

Seefeldt and Arp (187) have isolated membrane-boundAzotobacter vinelandii hydrogenase. The protein is com-posed of two subunits (Mr = 67,000 and 31,000) and contains0.7 g-atom of nickel and 6.6 g-atoms of iron per enzymemolecule. The enzyme from Azotobacter chroococcum hasnot been purified; however, it also was suggested to containnickel by Partridge and Yates (151). These conclusions arebased on the ability of chelating agents in the medium toprevent hydrogenase activity, whereas nickel addition to themedium restored the enzyme activity (151). Chelating agentshave also been shown to inhibit, in a nickel ion-reversiblemanner, the hydrogenases of Azospirillum brasilense,Azospirillum lipoferum, and Derxia gummosa (154). Thus,based on these data all aerobic nitrogen-fixing bacteria mayexhibit nickel-containing hydrogenases.

Other microorganisms. Hydrogenase has been purifiedfrom the anaerobic rumen bacterium Wolinella succinogenes(formerly Vibrio succinogenes) (210). This membrane-boundprotein is similar to those in aerobic cells described above.The protein possesses two subunits of Mr = 60,000 and30,000 and contains 1 g-atom of nickel per enzyme molecule.A second, nickel-containing hydrogenase also has beenisolated from this bacterium and shown to be reactive with2,3-dimethyl-1,4-naphthoquinone, a menaquinone analog(lla). EPR spectroscopic investigation of the enzyme en-riched in 33S indicated hyperfine interaction between para-magnetic nickel and sulfur, providing evidence for the pres-ence of a sulfur ligand.

In contrast to most of the microorganisms describedabove, Escherichia coli contains several different nickel-containing membrane-bound hydrogenases (22). Oneisoenzyme from anaerobically grown Escherichia coli re-cently was shown to possess two large subunits (Mr =64,000) and two small subunits (Mr = 35,000) and to contain12 g-atoms of iron and 0.6 g-atom of nickel per enzymemolecule (Mr = 200,000) (177). A second isoenzyme fromanaerobic cells was solubilized by using trypsin digestion.The solubilized protein derivative was made up of twopolypeptides (Mr = 61,000 and 30,000) with 12.5 g-atoms ofiron and 3.1 g-atoms of nickel per protein molecule (Mr =180,000) (23). Evidence for a third isoenzyme in anaerobi-cally grown Escherichia coli has also been reported, one thatis active in the formate hydrogen-lyase reaction (176). Form-ate-dependent growth of Escherichia coli is known to requirenickel (231). As in the case of anaerobic cultures, thehydrogenase of aerobically grown Escherichia coli alsoappears to contain nickel (84). Demonstration of multiplenickel-containing hydrogenases in Escherichia coli helps toillustrate the widespread nickel dependence among microor-ganisms, at least among those species participating in hydro-gen metabolism.

Further characterization of the nickel-containing hydrog-enases will be facilitated by using hydrogen uptake mutantswhich are defective in some aspect of nickel metabolism.The premier microorganisms for such studies areAlcaligenes eutrophus, Escherichia coli, and Bradyrhizo-biumjaponicum. The genetics of hydrogenase in Alcaligeneseutrophus has recently been summarized (73); several typesof mutants are characterized, but none is known to involvenickel. Two reports have appeared that describe hydrogenuptake mutants of Escherichia coli in which the deficiency isovercome by nickel (217, 226). Similar mutants are alsoknown in Bradyrhizobium japonicum (128).

In conclusion, however, it should be noted that not alluptake hydrogenases contain nickel. For example, uptakehydrogenases from Clostridium pasteurianum (2) andAcetobacterium woodii (164) do not contain this metal ion.

Nickel-Containing Methylcoenzyme M Reductases fromMethanogenic Bacteria

Methyl coenzyme M reductase, an essential enzyme foundin all methanogenic bacteria, possesses a nickel-containingcofactor named F430. Characterization of F430 and of its rolein the methyl coenzyme M reductase enzyme is discussedbelow.

Characterization of F430. In 1978, Gunsalus and Wolfereported the presence of a yellow, nonfluorescent compoundin heat-treated cell extracts of Methanobacterium ther-moautotrophicum strain AH (82). At the time, no role couldbe assigned to this chromophore, and it was termed factor

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COOCH3I3

COOH

A BFIG. 3. Structure of F430, a nickel-containing tetrapyrrole. (A) Structure of the methanolysis produce of F430 as deduced from nuclear

magnetic resonance spectroscopic analysis (159). (B) Native structure of the coenzyme.

F430 because it possessed an intense absorbance maximum at430 nm. Shortly thereafter, Thauer and co-workers observeda nickel requirement for growth of the Marburg strain ofMethanobacterium thermoautotrophicum (184). The resultsfrom these two experimental approaches converged in 1980when both groups, independently and simultaneously, re-ported that factor F430 contained nickel (53, 220). By usingthe radioisotope 63Ni, Diekert et al. (53) observed that 70%of the radioactivity taken up by the methanogen cells couldbe extracted and purified with factor F430, whereas Whitmanand Wolfe (220) demonstrated the presence of nickel inpurified F430 obtained from heat-treated Methanobacteriumbryantii cells by subjecting the chromophore to neutronactivation analysis. Other methanogens, includingMethanobrevibacter smithii, Methanosarcina barkeri,Methanococcus vannielii, and Methanospirillum hungatei,soon afterwards were also found to require nickel for growthand to possess factor F430 (54). To date, this pigment hasbeen found only in methanogens, and all methanogenspossess the nickel-containing factor F430.The structure of the nickel chromophore was examined by

growing methanogen cells in the presence of radioactivelylabeled precursors and testing the extracted F430 forbiosynthetic incorporation. Labeling patterns indicated that8 mol of either succinate (51) or 5-aminolevulinic acid (52)was incorporated per mol of F430. These results providedcompelling evidence for a tetrapyrrolic structure of thiscompound. The biosynthetic pathway for this chromophorewas further elucidated by the demonstration that radiola-beled uroporphyrinogen III could serve as a precursor forthe biosynthesis of F430 and that in the absence of nickel theuroporphyrinogen III levels increased in the cells (77).Additional labeling studies indicated that two methionine-derived methyl groups were present in F430 (96), similar tothe methylation patterns for the tetrapyrroles of sirohemeand vitamin B12. Indeed, sirohydrochlorin, a precursor in thesynthesis of the latter compounds, can be metabolized tofactor F430 by cell-free extracts of Methanobacteriumthermoautotrophicum (135). In an elegant collaborativestudy, Thauer, Eschenmoser, and colleagues deduced thestructure for this nickel-containing macrocycle (159).Methanobacterium thermoautotrophicum (strain Marburg)was grown in the presence of either specifically mono-13C-labeled 5-amino-levulinic acid (with the label at carbons two

through five) or with [methyl-13C]methionine. After cellgrowth, the F430 was extracted with perchloric acid, purified,and subjected to methanolysis and 13C-nuclear magneticresonance spectroscopic analysis. The pentamethyl esterstructure deduced by Pfaltz et al. (159) is shown in Fig. 3A.In Fig. 3B is shown the nonmethylated, pentaacid structurefor F430. This novel tetrapyrrole combines elements of boththe corrin and porphyrin ring systems and has been termed acorphin. The F430 tetrahydrocorphin possesses a uro-porphinoid ligand structure in which one acetamide sidechain is fused with a ring carbon to form a lactam, and apropionic acid side chain is cyclized to form a six-memberedcarbocyclic ring. The absolute configuration in rings a and brecently has been established (67).

Native F430 which had not undergone methanolysis wassuggested by other investigators to possess additional pe-ripheral components. Keltjens et al. (105-107) proposed thatseveral forms of F430 were present in the cell, includingderivatives containing bound coenzyme M (thioethane-sulfonate, a coenzyme found in all methanogenic bacteria)and 6,7-dimethyl-8-ribityl-5,6,7,8-tetrahydrolumazine. How-ever, these structural adducts could be artifacts generated byextracting F430 from whole cells under harsh conditions suchas high temperature or low pH. Indeed, several thermaldenaturation products of F430 and an oxidized form of F430,termed F56 for its absorbance maximum, have been struc-turally characterized (54, 160). By using mild extractionconditions, a single structural form of the corphin wasisolated from methanogen cells and was shown to be free ofperipheral components (89, 122). Additional evidence dem-onstrating the absence of coenzyme M was provided bygrowing Methanobacter ruminantium in the presence of[2-14C]coenzyme M and establishing the absence of radio-activity in the purified chromophore (94). Thus, the com-plete F430 structure is the penta-acid corphin shown in Fig.3B.

F430 exists in both protein-free and protein-bound forms(89, 122). The visible spectrum of protein-bound F430 isaltered when compared with the free chromophore; i.e., theabsorbance maximum is shifted to 418 to 422 nm and ashoulder is present at 445 nm (66, 132). The extinctioncoefficients at 430 nm for free F430 and at 420 nm for boundF430 are nearly identical and equal to 23,000 M-1 cm-1 pernickel molecule (57, 89). The total F430 content of cells (57)

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and free/bound ratio depends on the nickel content of themedium (15). At low nickel concentrations most of the F430is protein associated, whereas the protein-free F430 predom-inates at higher nickel concentrations (e.g., 1 ,uM). Whencells growing in nickel-rich media are down-shifted to nickel-deficient conditions, the protein-free F430 is converted to theprotein-bound form, suggesting a precursor-product rela-tionship (15). The chromophore can be dissociated from theprotein by using several methods, e.g., subjecting the en-zyme to a freeze-thaw cycle in 1 M NaCl, treatment with80% ethanol containing 2 M LiCl, and HC104 addition toadjust to pH 2. The structure of the chromophore extractedfrom the protein is identical to the protein-free state of F430(89, 122).Methyl coenzyme M reductase-bound F430. In a key publi-

cation from R. S. Wolfe's laboratory, the protein-boundform of F430 was identified as the chromophore of the methylcoenzyme M reductase enzyme (65). This enzyme is anessential and abundant protein in methanogenic bacteria,where it catalyzes the final reduction step in methane for-mation (equation 3) (66, 83). F430 is likely to participate inthis reduction and should be considered a coenzyme of themethyl coenzyme M reductase.

CH3-S-CH2CH2SO3- + 2H++ 2e -* HS-CH2CH2SO3-+ CH4(3)

methyl coenzyme M coenzyme M

In addition to the methyl coenzyme M reductase enzyme,three accessory proteins, hydrogen, component B (7-mercaptoheptanoylthreonine phosphate [145]), flavin ade-nine dinucleotide, Mg2+, and ATP are required to carry outthis reaction (136). Alternatively, a simplified in vitro assayfor methyl coenzyme M reductase has recently been de-scribed which requires the single reductase protein, compo-nent B, cobalamin, and dithiothreitol or SnCl2 as electrondonor (16). Although rigorous anaerobic conditions must bemaintained in the assay, methyl coenzyme M reductase isstable to oxygen and has been purified from severalmethanogens.The most intensively studied methyl coenzyme M

reductase enzyme is that isolated from Methanobacteriumthermoautotrophicum AH (83). This enzyme possesses ana2P2'Y2 structure composed of subunits with Mr = 68,000,45,000 and 38,500 and contains 2 mol of coenzyme F430, 2mol of coenzyme M, and an unknown amount of componentB per Mr = 300,000 (65, 107, 146). These coenzymes are notcovalently attached to the protein, nor are they covalentlybound to each other (88, 89). Hartzell and Wolfe (88) havesuccessfully reconstituted active enzyme from isolatedmethyl coenzyme M reductase subunits, exogenous F430,and methyl coenzyme M. Further, they have demonstratedthat F430 is probably associated with the largest subunit ofthis enzyme.The nickel center in enzyme-bound and protein-free F430

has been analyzed by EXAFS spectroscopy, a techniquewhich provides information on the type and number of nickelligands and the distances of these atoms from nickel. Eids-ness et al. (64) reported that the methyl coenzyme Mreductase-bound nickel is coordinated by five or six ligandsat a uniform distance of 0.209 nm. These ligands probablyinclude the four pyrrole nitrogen atoms and one or two axialoxygen or nitrogen atoms. Furthermore, a salt-extractedprotein-free form of F430 was found to possess a similarnickel coordination sphere. In contrast, the nickel of heat-extracted and other free F430 forms may be only four-coordinate, with nitrogen ligands at both 0.191 to 0.192 nm

and 0.21 to 0.214 nm (49, 64, 185). Eidsness et al. (64)speculate that the axial nickel coordination sites in theenzyme-bound F430 may be involved in catalysis.The protein-free F430 nickel ion is in the +2 oxidation state

(159) and cannot be detected by EPR spectroscopic meth-ods. However, a spectrum has been obtained forhexachloroiridate-oxidized F430 (J. Lancaster, personal com-munication in reference 178) and for dithionite-treated F430(8), consistent with nickel(III) and nickel(I) redox states,respectively. Alternatively, the low-intensity spectra ofthese preparations may arise from a contaminant such as anF430 degradation product. Very recently, Jaun and Pfaltz(100a) reported reduction of F430 pentamethylester dissolvedin tetrahydrofuran or dimethylformamide by using sodiumamalgam. The reduced species gave rise to an EPR spectrum(g = 2.250, 2.074, 2.065) that was consistent with a squareplanar nickel(I) complex. Albracht et al. (8) have observed apossibly related axial EPR spectrum (Table 3) attributed to S= 2 in octahedral geometry associated with purified prepa-rations of methyl coenzyme M reductase. The in vitro signalwas substoichiometric (3 to 16% of the F430 nickel) and wassuggested to represent only the active enzyme molecules inthe preparation. The novel spectrum associated with methylcoenzyme M reductase was also observed within intactwhole cells. These results may indicate that the activeenzyme contains F430 possessing a nickel(I) redox state. Theobservation of octahedral geometry is consistent with theEXAFS spectroscopy results, suggesting six-coordinate ge-ometry, discussed above.Resonance Raman spectroscopy has been used to charac-

terize the electronic transitions of protein-free F430 (albeit ofa thermally extracted, non-native form) (178). Three intensespectral features were observed at 1,628, 1,562, and 1,530cm-1, whereas several weaker spectral bands were foundbelow 1,500 cm-'. The intense features have been tenta-tively assigned to C=O, C=C, and C=N vibrations, and thelow-frequency bands probably include nickel-ligand interac-tions (178). This method also may prove useful in thecharacterization of protein-bound coenzyme; however, noreports of such studies have yet appeared.The role of the bound coenzyme F430 in methyl coenzyme

M reduction is unclear; nevertheless, several models can besuggested for the enzyme mechanism. F430 may function asa methyl carrier, somewhat analogous to the methylcorrinoid required for methionine biosynthesis (204). Thus,methyl coenzyme M may react with enzyme-activated F430to yield a methyl group axially bound to the F430 nickel. Thispotential intermediate may then cleave, either homolyticallyor heterolytically, to eventually yield methane. Alterna-tively, F430 may act as a hydride transfer agent and behavesimilarly to Raney nickel, a well-studied inorganic catalyst(92). Finally, a nickel-sulfur coordination during catalysiscannot be ruled out. Such a sulfonium ionlike axial associa-tion between methyl coenzyme M and F430 would promotedisplacement of the methyl group from the methyl coenzymeM thioether. These and other models for the action of methylcoenzyme M reductase might be tested by attempting to trapand characterize the intermediate catalytic states of thisenzyme. For example, the EPR studies of Albracht et al. (8)could perhaps be extended by using freeze-quench tech-niques to study the enzyme under catalytic conditions.

Nickel-Containing CO Dehydrogenases

Carbon monoxide (CO) is metabolized by a wide range ofmicroorganisms, many of which can assimilate CO into

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TABLE 4. Nickel-containing carbon monoxide dehydrogenases

Microorganism Evidence Subunit Mr Metal and cofactor contentb Reference(s)for Nia

Acetogenic bacteriaClostridium thermoaceticum p 78,000, 71,000 1.7 Ni, 1.3 Zn, 10.8 Fe/155,000 56, 62, 163Acetobacterium woodii p 80,000, 68,000 1.4 Ni, 1.0 Mg or Zn, 9 Fe/153,000 55, 167

Methanogenic bacteriaMethanosarcina barkeri p 92,000, 18,000 2 Ni, 30 Fe/232,000 113, Krzycki et al.,

unpublished dataMethanobrevibacter arboriphilus r,p NRC NR 85Methanosarcina thermophila p 89,000, 71,000, 3.6 Ni, 25 Fe, 1.2 Co, 6.1 207a

60,000, 58,000, Zn/297,00019,000

Other microorganismsRhodospirillum rubrum c NR NR 31Clostridium pasteurianum r,c NR NR 58, 61a The evidence for nickel in these enzymes includes the following: p, analysis of purified enzyme; r, nickel required for CO dehydrogenase activity; c,

comigration of 63Ni and enzyme activity in gels.b Gram-atoms of metal per Mr of the smallest enzyme unit.c NR, Not reported.

cellular carbon and use it as an energy source (130, 209).Aerobic CO-oxidizing bacteria include various species ofPseudomonas, Alcaligenes, Bacillus, Arthrobacter, Azoto-monas, and Azotobacter (130). These carboxydotrophicbacteria possess a molybdopterin-iron-sulfur-flavin-containing enzyme (nickel is not present) which has beentermed carbon monoxide:acceptor oxidoreductase and car-ries out equation 4 (130)

CO + H2O0i CO2 + 2H+ + 2e- (4)

Certain anaerobic bacteria carry out a similar reaction (50);however, a very different enzyme is utilized and generallyreferred to as CO dehydrogenase. The CO dehydrogenasesof several acetogens, methanogens, and other anaerobeshave been documented as nickel-containing enzymes (Table4). The evidence for the presence of nickel in these enzymesand the suggested roles for CO dehydrogenases are dis-cussed below.

Acetogenic bacteria. In 1978, Clostridium thermoaceticumand Clostridium formicoaceticum were each reported topossess highly active CO dehydrogenase (59). By use of 63Ni

* Carbon monoxide dehydrogenase

co CH3

co2 2 +2e

CH3A CO

Coenzyme A

FIG. 4. Simplified scheme depicting the role of carbon monoxidedehydrogenase (CODH) in acetogenesis (223, 224). The key reactionof this nickel-containing enzyme is the carbon-carbon bond forma-tion between two one-carbon precursor molecules.

in the growth medium, the partially purified CO dehydroge-nase from Clostridium thermoaceticum was shown to con-tain nickel (56, 62). More recently, this enzyme has beenpurified to homogeneity and shown to consist of twosubunits with Mr = 78,000 and 71,000 occurring in an cA33structure containing 2 g-atoms of nickel, 1 g-atom of zinc,and 11 g-atoms of iron per active enzyme dimer (Mr =155,000) (163). Nickel was also required for production ofCO dehydrogenase in Acetobacterium woodii (55). Thepurified enzyme from this microorganism also containsnickel and has properties similar to the Clostridiumthermoaceticum enzyme (167).CO dehydrogenase plays a central metabolic role in

acetogenic bacteria (123, 223). Drake et al. (63) purified fivecomponents from Clostridium thermoaceticum which arerequired for synthesis of acetate from pyruvate andmethyltetrahydrofolate and demonstrated that one of thefractions includes CO dehydrogenase activity. More re-cently, it was shown that acetyl coenzyme A was synthe-sized by the CO dehydrogenase and helper enzymes in thepresence of methyltetrahydrofolate, coenzyme A, and eitherCO (93) or carbon dioxide plus hydrogen (156). The in vivofunction of this enzyme recently has been elucidated byPezacka and Wood (157, 158) and Ragsdale and Wood (168).A simplified scheme is provided (Fig. 4) to demonstrate theimportant role of CO dehydrogenase in acetogenic bacteria.The enzyme carries out a carbonylation reaction in which acarbon-carbon bond is established between an enzyme-bound methyl group and a bound CO group. The methylgroup is derived from a corrinoid protein and the CO groupis derived from CO2 or directly from CO. The biologicalreaction is similar to the metal-promoted chemical synthesisof acetate from methyl iodide and CO, in the well-knownMonsanto process (71). In the cell, however, the acetylgroup is first transferred to coenzyme A rather than beingdirectly hydrolyzed to acetate. Because of the mechanism ofaction for this enzyme, Wood et al. (223) have suggested thatthe enzyme be renamed acetyl coenzyme A synthase. Thereactions carried out by this enzyme represent a new path-way for autotrophic growth (123, 223, 224, 224a) and as suchcan operate in Clostridium thermoaceticum to supportgrowth on carbon monoxide or hydrogen plus carbon dioxide(108).

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A putative nickel cofactor was extracted from 63Ni-labeledCO dehydrogenase with perchloric acid (163). The radioac-tivity labeled nickel fraction was chromatographed on acolumn of Sephadex G-25 and found to elute as a largecomplex (Mr > 1,350) rather than with an NiCl2 standard(163). No further characterization of the isolated nickelcofactor has been reported.The nickel center in the intact CO dehydrogenase has been

extensively studied by spectroscopic techniques. Thedithionite-free enzyme was found to exhibit a weak EPRspectral signal with g values of 2.21, 2.11, and 2.02 which isprobably derived from paramagnetic nickel(III) (166). Afterreacting the enzyme with either CO or CO2 under anaerobicconditions, EPR spectroscopy revealed a novel signal with gvalues of approximately 2.07 and 2.03 (165) (Table 3). Thebacteria were grown in 61Ni-enriched medium (nuclear spin= 3/2 for 61Ni) and the purified enzyme was again examinedby EPR spectroscopy. The observation of 6tNi hyperfine-induced broadening confirmed that the enzyme-bound nickelcontributed, at least in part, to the novel signal (166).Spectral broadening was also observed with 13CO (nuclearspin = 1/2) (166) and with 57Fe-enriched enzyme (169),demonstrating that the CO, carbon, and enzyme-bound ironand nickel are all spin coupled in a complex which isresponsible for the EPR spectrum. Some preparations ofCO-treated enzyme exhibited two superimposed EPR spec-tra, the above signal and a g = 2.05 signal (168, 169). Thebinding of coenzyme A converted the g = 2.05 signal to theinitially described spectrum, consistent with coenzyme Ainteraction near the active site nickel (168, 169). Furtherstudies are needed to characterize this complex metal-loenzyme active site.Methanogenic bacteria. Many of the methanogenic bacte-

ria have been shown to possess CO dehydrogenase, andrecently a dual role for the enzyme was proposed (50, 230).Biosynthetically, CO dehydrogenase is thought to functionby making acetyl coenzyme A during Methanobacteriumthermoautotrophicum autotrophic growth (196, 197), thesame as the acetogen enzyme. Thus, a methyl group from

CH3COOH

C02 * 2H+ 2e

CH4

FIG. 5. Scheme depicting the postulated role for carbon monox-ide dehydrogenase (CODH) in the methanogen acetoclastic reaction(50, 230). The nickel-containing enzyme cleaves the carbon-carbonbond of acetate. Electrons released during carbon monoxide oxida-tion are presumably utilized in the methyl coenzyme M (CoM)reduction step to form methane.

CH3-CoM

the methane bioenergetic pathway may be coupled to areduced form of carbon dioxide and coenzyme A to generateacetyl-coenzyme A, which is then utilized for further bio-synthesis. Second, a catabolic role of the enzyme has beenpostulated in certain methanogens which grow with acetate.These acetoclastic methanogens metabolize acetate to meth-ane and carbon dioxide, in an energy-yielding pathway (230).CO dehydrogenase is involved in this reaction (112, 142),presumably by a reversal of the biosynthetic reaction de-scribed above (Fig. 5). The electrons required for methylcoenzyme M reduction are apparently derived from oxida-tion of the protein-bound CO (142). The reversibility ofpurified enzyme preparations has not been reported; never-theless, the methanogen CO dehydrogenase may operate inthe cell bidirectionally to satisfy either biosynthetic or en-ergy demands.

Progress has been achieved recently in the purification andcharacterization of methanogen CO dehydrogenase.Methanobrevibacter arborphilicus was grown auto-trophically in the presence of 63Ni (85) and the radioactivitywas copurified (21-fold) with the enzyme. In addition, Meth-anosarcina barkeri CO dehydrogenase was isolated fromacetate-grown cultures and the enzyme was also shown tocontain nickel (113). This enzyme has an x42N structure withsubunit Mr = 92,000 and 18,000, respectively. Although nonickel EPR signals were detected, the Methanosarcinabarkeri CO dehydrogenase was shown to contain 2 g-atomsof nickel and 30 g-atoms of iron per enzyme molecule (J. A.Krzycki, L. E. Mortenson, and R. C. Prince, Abstr. Annu.Meet. Am. Soc. Microbiol. 1986, I51). CO dehydrogenasewas purified 10-fold from acetate-grown Methanosarcinathermophila strain TM-1. This CO dehydrogenase may havefive differently sized subunits and is reported to contain 3.6g-atomns of nickel, 25 g-atoms of iron, 1.2 g-atoms of cobalt,and 6.1 g-atoms of zinc per enzyme molecule (Mr = 297,000)(207a). Further understanding of the roles for CO dehydro-genase in methanogens will require more detailed studies ofpurified enzymes.

Other bacteria. A nickel requirement for CO dehydroge-nase formation was first observed in Clostridiumpasteurianum (58). When the organism was grown in thepresence of 63Ni, the CO dehydrogenase activity was foundto correlate with a radioactive band after gel electrophoresis(61). Unfortunately, no more information is available sincethe nickel center of the enzyme is labile and the protein hasnot been isolated. The role of CO dehydrogenase in Clos-tridium pasteurianum is also unclear.CO dehydrogenase has been partially characterized from

two purple, nonsulfur phototrophs. The enzyme was purified600-fold from Rhodospirillum rubrum grown underanaerobic, phototrophic conditions (31). The cells werefound to take up 63Ni very poorly from the medium; how-ever, trace amounts of the radioisotrope did comigrate withthe enzyme on native gels. The authors were not convincedthat this enzyme contains nickel and further experiments areneeded to answer the question (31). In contrast to the solubleenzymes described above, the Rhodocyclus gelatinosus (for-merly Rhodopseudomonas gelatinosus) CO dehydrogenaseis membrane associated (216). This microorganism exhibitsrapid growth in the dark under 100% CO by obtaiting energythrough CO oxidation (216). Since the enzyme has not beenisolated, the nickel content is not known. Moreover, EPRspectroscopy provided no evidence for nickel in oxidized orreduced Rhodocyclus gelatinosus membrane samples (R.Uffen, personal communication).The first evidence for anaerobic oxidation of CO was

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TABLE 5. Summary of nickel transport in microorganisms

Mechanism of nickel Species Km Vmax (pmol min-' Reference(s)transport mg of dry wtV')

Mg2+-specific system Saccharomyces cerevisiae 0.5 mM 0.5 76Neurospora crassa 0.29 mM 15 131Escherichia coli NDa ND 100, 218Enterobacter aerogenes ND ND 218Bacillus megaterium ND ND 218

Ni2+-specific system Alcaligenes eutrophus ND ND 200Methanobacterium bryantii 3.1 ,uM 24 99Anabaena cylindrica 17 nM 0.37 34Bradyrhizobium japonicum 26 ,uM ND Stults and Maier, unpublished dataClostridium thermoaceticum ND 4 Lundie and Drake, unpublished data

Unknown Azotobacter chroococcum ND ND 151Phaeodactylum tricornutum ND ND 188

a ND, Not determined.

reported by Yagi in 1958 (228), using cell extracts ofDesulfovibrio desulfuricans. Some of the sulfate-reducingbacteria are capable of autotrophic growth, and it nowappears likely that the CO dehydrogenase-dependent acetyl-coenzyme A pathway is used (97). Nevertheless, CO dehy-drogenase has not been purified from any sulfate-reducingbacterium, and no requirement for nickel has been demon-strated.

OTHER MICROBIAL ROLES FOR NICKEL

Three types of algae, Chlorella vulgaris (26), Chlorellaemersonii (189), and an Oscillatoria species (211), requirenickel for growth. These microorganisms possess urease;however, the nickel dependence is unrelated to this enzyme(189, 211) and may be due to a nickel-containing hydroge-nase. Further studies are required to substantiate this sug-gestion.

Subsequent to the initial report demonstrating a nickelrequirement for methanogens (184), nickel was establishedas a component of the methanogen enzymes methyl coen-zyme M reductase (65), carbon monoxide dehydrogenase(85, 113), and hydrogenase (80). In addition to these knownactivities, nickel may perform other functions. Methano-bacterium bryantii was found to undergo lysis in nickel-depleted media lacking ammonia (98). Thus, nickel may playa role in cell wall biosynthesis or stabilization in this micro-organism. The cytoplasmic membrane of Methanospirillumhungatei contains significant nickel content, accounting for0.16% of the membrane dry weight (193). The sheath sur-rounding the same methanogenic cell also contains apprecia-ble nickel (193), even after extensive dialysis. However, therole for sheath and membrane-bound forms of nickel in thecells is not known.

In a strain of Bacillus cereus, a nickel-containing pigmentwas mistakenly reported to be involved in endospore forma-tion (208). At present there is no indication that nickel playsany positive role in the physiology of this bacterium. Rather,a low-molecular-weight compound produced by this microbechelates nickel and forms large, dark-green complexes whichmay serve in a detoxification role (J. Ormerod, personalcommunication).

MECHANISMS OF NICKEL UPTAKE

The demonstration of widespread nickel requirementsamong microorganisms has sparked interest in the area ofcellular nickel transport (Table 5). To examine this problem,

extensive use has been made of the p-emitting nickel isotope63Ni. In these studies, great care must be taken to distinguishnickel transport from simple nickel uptake, i.e., nickelbinding to cell walls and extracellular components. Forexample, isolated Bacillus subtilis cell walls were found byatomic absorption analysis to bind 18 different metals (27).Although only intermediate amounts of nickel were bound, itwas strongly absorbed. Initial nickel binding of this type maybe a prerequisite of the actual transport of nickel into the cellin some microorganisms (103).One of the earliest studies to detail nickel transport

involved the yeast Saccharomyces cerevisiae (76). Severalmetals are taken up by the cells in the affinity series Mg2+,Co2+ > Zn2+ > Mn2+ > Ni2+ > Ca2I > Sr2+. Metalincorporation into yeast cells is energy dependent as shownby enhanced nickel transport in the presence of glucose (76).As with the yeast cell, nickel transport in Escherichia coli(100, 218), Enterobacter aerogenes (formerly Aerobacteraerogenes) (218), Bacillus megaterium (218), and Neuros-pora crassa (131) may also occur via a magnesium-specific,energy-dependent mechanism. Phaeodactylum tricornutumalso possesses an energy-dependent nickel uptake system;however, this system is stimulated by phosphate (188). Incontrast to the free metal ion uptake systems, Bacillussubtilis possesses an energy-dependent system which trans-ports nickel as a citrate complex (221). The magnesium-citrate complex is preferentially transported by the cell, andthe role of the transport system may be for citrate utilization.Finally, Azotobacter chroococcum differs from each of theabove microorganisms by accumulating nickel in an energy-independent manner (151).The first high-affinity nickel transport system was de-

scribed by Tabillion and Kaltwasser (200) for two strains ofAlcaligenes eutrophus. Autotrophically grown cells accumu-lated nickel 280-fold over the concentration in the medium,with optimal growth occurring at 0.3 pLM nickel in the culturemedium. Nickel transport was energy dependent and washighly specific; e.g., there was only slight inhibition by Zn2+,Co2+, Mn2+, and Cu2+. The nickel taken up by the cell ispredominantly incorporated into two cellular hydcrogenases(74).Methanogenic bacteria have a pronounced requirement

for nickel ion because of their multiple nickel-containingenzymes. Thus, it came as no surprise when Jarrell andSprott (99) established that a high-affinity nickel transportsystem was present in Methanobacterium bryantii. Nickeltransport was highly specific as shown by a lack of interfer-ence by other metal ions, except cobalt. The Km of the

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uptake system for nickel was 3.1 ,uM, similar to the optimalnickel concentration required for methanogen growth, andthe maximum rate of nickel transport is 24 pmol min-' mg ofdry cells-'. Nickel accumulation is inhibited by nigericin,monensin, and gramicidin, and transport is stimulated by anartificial pH gradient. The authors concluded that nickelaccumulation is coupled to proton movement in this micro-organism (99). Similarly, nickel uptake in Methanospirillumhungatei requires a proton flux (192).

In a very recent publication, Campbell and Smith charac-terized the nickel transport system of the cyanobacteriumAnabaena cylindrica (34). Nickel transport was unaffectedby the presence of other metal ions. An extremely low-affinity constant for nickel was demnonstrated (Km = 17 nM)and the cells were shown to concentrate nickel 2,700-fold.Nickel transport was quite slow, however, with a Vmax of0.37 pmol of nickel min-1 mg of dry cells-1. The authorssuggested that a slow transport rate may be necessary toprevent nickel toxicity. A carrier-facilitated transport sys-tem was proposed which is dependent on the membranepotential. Inhibition of nickel transport was observed in thepresence of carbonyl cyanide m-chlorophenylhydrazone andsimilar uncouplers, as well as with dicyclohexylcarbo-diimide, an adenosine triphosphatase inhibitor. In additionto the transport system, the cells exhibited rapid extracellu-lar binding of nickel. This organism is known to have both anickel-dependent hydrogenase and urease (34).

In unpublished work (L. W. Stults and R. J. Maier,personal communication), heterotrophically grown Brady-rhizobium japonicum appeared to possess a nickel transportsystem with a Km of 26 ,uM. Nickel transport was unaffectedby magnesium, cobalt, iron, or manganese; however, a10-fold excess of zinc or copper over nickel did result in a45% loss of uptake. Interestingly, a hydrogen uptake-constitutive (Hupc) mutant was found to accumulate 10-foldmore nickel and to transport nickel ion at five times the ratecompared with wild-type Bradyrhizobium japonicum. Hy-drogenase in this microbe is known to contain nickel (17, 86,195).Nickel transport has recently been examined in the

acetogen Clostridium thermoaceticum (L. L. Lundie andH. L. Drake, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985,K1). The nickel ion is exclusively incorporated into thenickel-containing carbon monoxide dehydrogenase. Twonickel uptake systems were detected; i.e., a slow transporter(4 pmol of nickel min-' mg of dry cells-') is saturated at 50,M nickel, whereas a second rapid system (580 pmol ofnickel min-' mg-') requires 5 mM nickel for saturation. Inthis high-affinity nickel transport system, as in others dis-cussed, divalent cations do not inhibit and the transport isenergy dependent.Many iron-dependent microorganisms synthesize and ex-

crete siderophores which complex ferric ion (140). Thebiosynthetic iron chelators fall into two general classes: thephenolate/catecholate type and the hydroxamate type (140).Hydroxamic acids are also known to avidly chelate nickel(which is why these compounds act as urease inhibitors [30,175]). Many nickel-requiring microorganisms producehydroxamate compounds (5, 34, 140) and a role in nickeltransport may be suggested for such chelators. As describedearlier, for example, Bacillus subtilis can transport nickel asa nickel-citrate complex (221). To examine the need fornickel chelators, Gloss and Hausinger (unpublished data)studied the spent culture medium from Methanobacteriumthermoautotrophicum which had been grown in the absenceof added nickel. It was hoped that nickel-deficient growth

conditions would amplify the level of any secreted chelators.However, no evidence for a phenolate/catechol type ofcompound was obtained, and only trace levels of ahydroxamate compound were detected in the culture fluid. Itmnust be kept in mind that nickel(II) ion is quite soluble, incontrast to the ferric hydroxide situation with a solubilityconstant of 10-38 M. Furthermore, the high-affinity nickeltransport systems discussed above were generally exa

nickel utilization by microorganismst · the most intensively examined urease is the plant enzyme...

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