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oxide species.This is followed by hetero-lytic cleavage of the OO bond, pro-ducing the elusive intermediate knownas compound I, formally an FeV spe-cies. The latter behaves as an extremelypowerful oxidant with a redox potentialprobably exceeding + 1 V. It has beenproposed to react directly with thesubstrate in a fast rebound mechanismto yield the hydroxylated product andregenerate the FeIII active site. How-ever, chemical modeling as well asstudies on related enzymes such ascatalases and peroxidases suggest thatthese species feature a high-valent[FeO]2+ core bound to a porphyrinradical cation rather than a genuineFeV moiety. Fascinating recent develop-ments in the chemistry of CYP450CAMinclude the crystallographic character-

ization of several of the intermediates inthe reaction cycle[3a] as well as thequantum-mechanical study of the reac-tion cycle with explicit inclusion of theprotein environment.[3b]

Recently, attention has been focusedon mononuclear NHI proteins, whichhave proven to be as versatile as hemo-proteins in oxygen-activation process-es.[4] However, a significant point ofconcern is whether the NHI enzymescould produce high-valent-iron inter-mediates in the absence of a porphyrin

ring acting as an electron reservoir. Theexistence of compound I-like intermedi-ates has been challenged on the basis ofcrystallographic studies which haveshown that the iron centers in NHIenzymes are always coordinated to in-nocent ligands that presumably can notbe ionized at the biologically relevantredox potential window. An attractivealternative to the production of high-valent intermediates is the direct reac-tion of FeIIIhydroperoxides to yieldhydroxylated or epoxidized products

(Scheme 2). Recent studies have shownthat low-spin and high-spin FeIII hydro-peroxides are activated for homolyticOO and FeO bond cleavage, respec-tively.[5] The deprotonated form, O22, isfound in side-on-bound high-spin h2-FeIIIperoxo complexes, which do notappear to be activated for direct reac-tion with substrates.[6] The direct reac-tion of low-spin FeIIIhydroperoxidecomplexes is, therefore, an alternativemechanism and has been proposed tooccur, for instance, in the oxidation of

DNA by the anticancer drug bleomy-

cin.[7]

FeIV

(or even FeV

) intermediateshave been frequently proposed in NHIenzyme mechanisms, but have not beendetected experimentally. This has in-spired synthetic chemists to attempt toprepare high-valent iron model com-plexes, leading to the first spectroscopiccharacterization of complexes featuringthe terminal [FeO]2+ and [FeN]2+ moi-eties[8]

The article by Mnck, Nam, Que,and co-workers is a significant advance-ment towards the preparation and char-

acterization of mononuclear high-valentnonheme iron centers.[1] In this study,they provide complete structural andspectroscopic characterization of the[FeO]2+ core in a nonheme environ-ment. The synthesis involved the reac-tion of the precursor [Fe(tmc)(OTf)2] (1;tmc= 1,4,8,11-tetramethyl-1,4,8,11-tet-raazacyclotetradecane, OTf=CF3SO3)

with iodosylbenzene (PhIO, an oxygen-atom-transfer reagent) in acetonitrile at40 8C to yield [Fe(tmc)(O)(CH3CN)]2+

(2). This species was characterized byabsorption, FT IR, and Mssbauer spec-troscopy, as well as by mass spectrome-try. Crystallization from CH3CN/Et2O at40 8C yielded the triflate salt 2(OTf)

2.

Its structure consists of a mononucleariron center in a distorted octahedralenvironmentwith a short terminal FeObond (1.646(3) , Figure 1). This bondis much shorter than those in bridgingoxo ions in dinuclear complexes featur-ing FeIV (1.805 ),[9] or the terminalFeO bond in a recently characterizedcomplex featuring the [FeO]+ fragment(1.813 ),[10] and is indicative of in-creased p-bonding within the terminal[FeO]2+ moiety. The short FeO bond

length is similar to those deduced fromEXAFS (extended X-ray absorptionfine structure) spectroscopic measure-ments on high-valent porphyrins andalso resembles the values based on theX-ray structural analysis of hemopro-teins featuring the [FeO]2+ core.[11]

This remarkable results of Mnck,Nam, Que, and co-workers provides awell-characterized model complex nec-essary for evaluating the feasibility ofhigh-valent ironoxo species in the re-action pathways of NHI enzymes. In

addition, these results allow studying thespectroscopic properties of the high-valent NHI oxo unit in the absence ofimpurities or other chromophores. Thisprovides a sound structural basis forfuture spectroscopic assignments of thisstructural motif and an in-depth under-standing of the relationship betweenelectronic structure, spectroscopy, andreactivity in these systems.

Biosynthesis of the FeCN Bond

in NiFe Hydrogenases

Hydrogen gas is an essential nutrientin virtually all forms of life that inhabitanaerobic environments, such as deep-sea hydrothermal vent sites, lake sedi-ments, and the intestines of animals. H2is produced by fermentative organismsin the oxidation of sugars and otherorganic molecules. The liberated elec-trons are transferred to protons throughan electron-transport chain reaction(which involves, among others, the Fe

Scheme 2. Important intermediates in thereactions of NHI enzymes.

Figure 1. Molecular structure of the [Fe(tmc)(O)(CH3CN)]2+ dication, derived from X-raycrystallography. The given values indicatebond lengths in . Hydrogen atoms are omit-ted for clarity.

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S protein ferredoxin) to produce H2 gas.This final key step is catalyzed by afamily of enzymes that are collectivelyknown as hydrogenases. Hydrogen gas isrecycled by other organisms in the samehabitat. These bacteria also containhydrogenases that catalyze the reversereaction, that is, the oxidation of H

2.

Although several different classes ofhydrogenases are known, the most com-mon of these enzymes contain both Niand Fe ions in their active center (NiFe-H2ase). The crystal structure of theNiFe-H2ase from Desulfovibrio gigaswas solved in 1995[12] and showed thatthe active site contains a heterodinu-clear NiFe site with two terminal cys-teine ligands at the Ni end (Figure 2). In

addition, there are two bridging cysteinefragments and an unidentified ligand(probably OH), which bridge to anearby Fe center. Recently, a detailedspectroscopic study has provided insightinto the electronic structure of the activecenter and of the reaction intermedi-ates.[13]

The most striking structural featureof the NiFe-H2ases involves the threeterminal diatomic molecules coordinat-ed to the Fe center (Figure 2). Thesediatomic species were identified byFTIR spectroscopy as being one COand two CN ligands.[14] This result wasquite surprising as both CO and CN arehighly toxic for biological systems be-cause of their high affinity for the activesites of many metalloenzymes (e.g. he-moproteins involved in cell respiration).CN and CO were also found in the

active sites of other hydrogenases. It hasbeen proposed that this highly unusualarrangement helps to keep the Fe centerin a low-spin low-valent state, whichmay be relevant to the function ofH2ases.

The identification of CN and COligands in the active site of metallopro-teins subsequently prompted the ques-tion of how these toxic species aredelivered to the active sites of theenzymes during the biosynthesis, as COand CN are not freely available inbiological systems. Bck and co-workershave provided an explanation for thisprocess in the NiFe-H2ases.[2] Using acombination of radio isotope labelingand mass spectrometry, they showedthat organic thiocyanates are a sourceof CN. These thiocyanates are formed

as a result of the interaction of two(HypF and HypE) out of seven proteinsinvolved in the maturation of the NiFeactive site. A detailed mechanism isoutlined in (Scheme 3): The first stepof the biosynthesis involves the forma-tion of a carbamoyl adenylate inter-

mediate by the reaction between carba-moyl phosphate (CP, a known inter-mediate in biosythetic pathways, e.g.,the urea cycle) and ATP on the HypFprotein. The CONH2 group is subse-quently transferred to the thiol group ofthe C-terminal cysteine residue on thesecond protein HypE. This protein thenperforms a complex set of unprecedent-ed reactions: it provides the site fordehydration of the carboxamido moietyin an ATP-dependent step to yield thecyanated cysteine (thiocyanate). Thisintermediate presumably carries theSCN residue to the active site and finallytransfers the electrophilic CN moiety toa nucleophilic iron center to form theFeCN unit.

Bck and co-workers explored thismechanism further with a series of

elegant model experiments to probethe key steps described above. Theyshowed that the dehydration of S-(n-decyl)thiocarbamate assisted by ethylpolyphosphate proceeds under mildconditions and yields the correspondingthiocyanate species in a way that resem-

Figure 2. Schematic representation of the ac-tive site of the NiFe-H2ase from D. vulgaris ob-tained by X-ray crystallography of the oxidized

protein. The fragment X identifies the thirdbridging ligand, which has not been identifiedbut is most probably an OH group. PDBcode 2FRV.

Scheme 3. Biosynthesis of the cyanide ligand in NiFe-H2ases: 1) formation of the carbamoyl ad-enylate on the HypF protein (F) involving an ATPPPi exchange reaction; 2) the CONH2 group istransferred to the C-terminal cysteine of the HypE protein (E); 3) and 4) ATP-assisted dehydra-tion involving an intermediate phosphorylation of the carbonyl oxygen atom followed by dephos-phorylation to yield a thiocyanate; 5) chemical transfer of the CN group to the active center ofthe NiFe-H2ase protein (LN represents the coordination sphere of the active site). ATP=adeno-sine triphosphate, ADP=adenosine diphosphate, AMP=adenosine monophosphate, Pi=phos-phate, PPi=pyrophosphate.

Highlights

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bles the natural system. By treatingphenyl thiocyanate with [(h5-C5H5)Fe-(CO)2Br] (FpBr) to generate [(h5-C5H5)Fe(CO)2CN] (FpCN) in goodyield, they showed that organic thiocya-nates could directly deliver CN to low-valent Fe centers under mild conditions.

The combination of experiments onthe natural protein system and on orga-nometallic model compounds have shedlight on a remarkable biosynthetic path-way. It was clearly demonstrated thatmetalCN units can be formed underbiological conditions in the absence offree CN. A viable mechanism for theorigin of these unusual ligands in theactive site of hydrogenases has thusbeen provided.

Conclusion

The two studies by Mnck, Nam,Que, and co-workers[1] and Bck and co-workers[2] are clear examples of how thechemical principles underlying compli-cated biochemical reactions can beclarified with the use of synthetic modelcompounds. Furthermore, they demon-strate how potentially toxic intermedi-

ates are controlled and/or avoided inbiological systems.

[1] J. U. Rohde, J. H. In, M. H. Lim, W. W.Brennessel, M. R. Bukowski, A. Stubna,E. Mnck, W. Nam, L. Que, Jr., Science2003, 299, 1037.

[2] S. Reissmann, E. Hochleitner, H. Wang,A. Paschos, F. Lottspeich, R. S. Glass, A.Bck, Science 2003, 299, 1067.

[3] a) I. Schlichting, J. Berendzen, K. Chu,A. M. Stock, S. A. Maves, D. E. Benson,B. M. Sweet, D. Ringe, G. A. Petsko,S. G. Sligar, Science 2000, 287, 1615;b) J. C. Schneboom, H. Lin, N. Reuter,W. Thiel, S. Cohen, F. Ogliaro, S. Shaik,

J. Am. Chem. Soc. 2002, 124, 8142.[4] E. I. Solomon, T. C. Brunold, M. I. Da-

vis, J. N. Kemsley, S. K. Lee, N. Lehnert,F. Neese, A. J. Skulan, Y. S. Yang, J.Zhou, Chem. Rev. 2000, 100, 235.

[5] a) N. Lehnert, F. Neese, R. Y. N. Ho, L.

Que, Jr., E. I. Solomon, J. Am. Chem.Soc. 2002, 124, 10810; b) N. Lehnert,R. Y. N. Ho, L. Que, Jr., E. I. Solomon,

J. Am. Chem. Soc. 2001, 123, 12802.[6] F. Neese, E. I. Solomon, J. Am. Chem.

Soc. 1998, 120, 12829.[7] a) F. Neese, J. M. Zaleski, K. L. Zaleski,

E. I. Solomon, J. Am. Chem. Soc. 2000,122, 11703; b) E. I. Solomon, A. Deck-er, N. Lehnert, Proc. Natl. Acad. Sci.USA 2003, 100, 3589.

[8] a) K. Meyer, E. Bill, B. Mienert, T.Weyhermller, K. Wieghardt, J. Am.Chem. Soc. 1999, 121, 4859; b) C. A.Grapperhaus, B. Mienert, E. Bill, T.Weyhermller, K. Wieghardt, Inorg.Chem. 2000, 39, 5306.

[9] a) M. Costas, J. U. Rohde, A. Stubna,R. Y. N. Ho, L. Quaroni, E. Mnck, L.Que, Jr., J. Am. Chem. Soc. 2001, 123,12931; b) H. F. Hsu, Y. H. Dong, L. J.Shu, V. G. Young, L. Que, Jr., J. Am.Chem. Soc. 1999, 121, 5230.

[10] C. E. MacBeth, A. P. Golombek, V. G.Young, C. Yang, K. Kuczera, M. P.Hendrich, A. S. Borovik, Science 2000,

289, 938.[11] a) J. E. Penner-Hahn, K. S. Eble, T. J.

McMurry, M. Renner, A. L. Balch, J. T.Groves, J. H. Dawson, K. O. Hodgson, J.

Am. Chem. Soc. 1986, 108, 7819; b) T.Wolter, W. Meyer-Klaucke, M. Muther,D. Mandon, H. Winkler, A. X. Traut-wein, R. Weiss, J. Inorg. Biochem. 2000,78, 117.

[12] A. Volbeda, M.-H. Charon, C. Piras,E. C. Hatchikian, M. Frey, J. C. Fonte-cilla-Camps, Nature 1995, 373, 580.

[13] S. Foerster, M. Stein, M. Brecht, H.Ogata, Y. Higuchi, W. Lubitz, J. Am.Chem. Soc. 2003, 125, 83.

[14] K. A. Bagley, E. C. Duin, W. Roseboom,S. P. J. Albracht, W. H. Woodruff, Bio-chemistry 1995, 34, 5527.

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