[2] TYROSYL RADICALS AND RNRs 21

oxygen species by dedicated enzyme systems (thioredoxin, glutaredoxin, and/or H-redoxin) that reduce the thiols during catalysis.

A c k n o w l e d g m e n t s

We thank Sabrina Bodevin, Per Siegbahn, and Rula Zain for valuable discussions, Annika Persson for help with Fig. 2, and Euan Gordon for linguistic help. Research in the authors' laboratory was sup- ported by grants from the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research, the Swedish Natural Science Research Council, and Carl Trygger's Foundation.

[2] Tyrosyl Radicals and Ribonucleotide Reductase

By MARC FONTECAVE and CATHERINE GEREZ

I n t r o d u c t i o n

DNA synthesis depends on a balanced supply of the four deoxyribonucleotides, which is achieved by reduction of the corresponding ribonucleoside diphosphates, NDPs, or triphosphates, NTPs. 1 This reaction is catalyzed by a fascinating family of radical metalloenzymes, named ribonucleotide reductases (RNRs). In contem- porary metabolism, at least three distinct classes of RNR are found, having in common the requirement for a transient cysteinyl radical in the active site during catalysis. 2-5 The difference among the three classes resides in the cofactors used to generate the radical.

Class I RNRs are strictly aerobic, found in all types of eukaryotes, several viruses, and a few prokaryotes, such as Escherichia coli. Protein R1, the large component, is the site where the reduction of ribonucleotides takes place. The electrons are provided by NADPH and transferred to R1 through the thioredoxin reductase/thioredoxin system. Protein R2, the small component, contains a stable tyrosyl radical essential for enzyme catalysis, as it serves to generate, through a long-range radical transfer reaction, the active cysteinyl radical in R1. 3 It also contains a nonheme diiron center, required for the oxygen-dependent generation of the tyrosyl radical. 6

1 p. Reichard, Annu. Rev. Biochem. 57, 349 (1988). 2 M. Fontecave, Cell. Mol. Life Sci. 54, 684 (1998). 3 B.-M. SjOberg, Struc. Bond. 88, 139 (1997). 4 A. Jordan and E Reichard, Annu. Rev. Biochem. 67, 71 (1998). 5 j. Stubbe and W. A. van der Donk, Chem. Rev. 98, 705 (1998). 6 j. Stubbe and E Riggs-Gelasco, Trends Biochem. Sci. 23, 438 (1998).

Copyright © 2002 by Academic Press. All rights of reproduction in any form reserved.

METHODS IN ENZYMOLOGY, VOL. 348 0076-6879102 $35.00

22 PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES [2]

Class II RNRs are ot or ct2 aerobic or anaerobic enzymes found in bacteria and archaea and defined as using adenosylcobalamin (AdoCbl) as a cofactor. 4'5'7 The enzyme facilitates homolysis of the C o - C bond of AdoCbl for generating the essential cysteinyl radical. 8

Class III RNRs are oxygen-sensitive c~2/~2 enzymes found in some facultative anaerobes, bacteriophages, and archaea. 4'7 The large component c~2 is the proper reductase. In its active form, it carries a glycyl radical absolutely required for catalysis. Binding of the substrate is supposed to trigger radical transfer to an adjacent cysteine in the active site. 9 The small component/~2 contains an iron sulfur center, which serves for the generation of the glycyl radical, a reaction requiring S-adenosylmethionine as a cofactor.

This article describes methods [light absorption and electron paramagnetic resonance (EPR) spectroscopy] for monitoring the various types of tyrosyl radicals in class I RNRs. These radicals are important targets for antiproliferative compounds.

P r e p a r a t i o n o f T y r o s y l R a d i c a l - C o n t a i n i n g C o m p o n e n t o f C l a s s I R i b o n u c l e o t i d e R e d u c t a s e f r o m D i f f e r e n t S p e c i e s

As described here, three types of tyrosyl radicals have been observed among a large number of purified reductases. R2 proteins from E. coli, Mycobacterium tuberculosis, and Arabidopsis thaliana may be considered as representatives of each of these classes.l°-13 The three proteins can be prepared from overexpressing E. coli strains, available in this laboratory, which have been transformed with the corresponding plasmids: pVNR2,14 pMtbR2,11,12 and pETR2,13 respectively.

Overexpression and Purification o f R2 Protein from E. coli (R2ec), A. thaliana (R2at), and M. tuberculosis (R2mO

The E. coli host strains used for overexpressing R2ec, R2at, and R2mt are K12, B834(DE3)pLysS, and BL21(DE3), respectively. 1°-14 Cells transformed

7 M. Fontecave and E. Mulliez, in "Chemistry and Biochemistry of B12" (R. Banerjee, ed.), p. 731. Wiley-Interscience, New York, 1998.

8 S. Licht, G. J. Gertin, and J. Stubbe, Science 271, 477 (1996). 9 D. T. Logan, J. Andersson, and B.-M. Sjfberg, Science 283, 1499 (1999).

10 B.-M. Sjrberg, S. Hahne, M. Karlsson, H. Jrmvall, M. Grransson, and B. E. Uhlin, J. Biol. Chem. 261, 5658 (1986).

11 F. Yang, G. Lu, and H. Rubin, J. Bacteriol. 176, 6738 (1994). 12 E. Elleingand, C. Gerez, S. Un, M. Kntipling, G. Lu, J. Salem, H. Rubin, S. Sauge-Merle, J. P.

Laulhbre, and M. Fontecave, Eur. J. Biochem. 258, 485 (1998). ~3 S, Sauge-Merle, J. P. Laulh~re, J. Coves, L. Le Pape, S. MEnage, and M. Fontecave, J. Biol. Chem.

2, 586 (1997). 14 C. Gerez, E. Elleingand, B. Kauppi, H. Eklund, and M. Fontecave, Eur. J. Biochem. 249, 401 (1997).

[2] TYROSYL RADICALS AND RNRs 23

with the corresponding plasmids are grown at 37 ° in LB medium supplemented with the appropriate antibiotics until an absorbance at 600 nm of 0 .4-0 .6 is reached. Then, protein expression is induced by the addition of 0.4-1 mM iso- propyl-/%D-thiogalactopyranoside (IPTG). About 4 hr later, cells are collected by centrifugation, resuspended in the appropriate buffer, and frozen in liquid nitrogen before storage at - 8 0 ° . All purification steps are performed at 4 ° .

The frozen cells are lysed by freeze-thawing rupture, and the total protein extract is recovered by a 90-min centrifugation at 45,000 rpm in rotor 60 TI (Beckman). DNA and rRNA are precipitated by the addition of 2% strepto- mycin sulfate and removed by centrifugation (20,000g for 30 min), and the su- pernatant is concentrated by ammonium sulfate precipitation [60% (R2ec and R2mt) or 50% (R2at) final saturation]. The pellet, recovered by centrifugation (20,000g for 30 min), is dissolved in a minimal volume of 50 mM Tris-C1, pH 7.6 (buffer A), containing 20% (v/v) glycerol (R2ec), 5% (v/v) glycerol (R2at), or 0.1 mM dithiothreitol (DTT) (R2mt). The subsequent chromatography steps are summarized in Table I. For R2ec and R2mt, as the first step is an anion- exchange chromatography, salts are first removed by gel filtration on a Sephadex G-25 column (R2ec) or by dialysis against buffer A for 5 hr with one buffer change (R2mt).

TABLE I PURIFICATION OF R2 PROTEINS FROM E. coli, A. thaliana, AND M. tuberculosis

Step R2ec R2at R2mt

First fractionation

Column a Buffer

Second fractionation

Column a

Buffer

DE52 Sephacryl S-100 HR Gradient: 0.15 to 0.3 M Buffer m b, 5% (V/V)

KPO4, pH 7 glycerol, 1 M KCI

Phenyl-Sepharose CL-4B

Wash: buffer A, b 1 M KCI Elution: buffer A b

DE52 Wash: 3 volumes buffer A b

with 0.1 mM DTT, 0.1 M KCI

3 volumes buffer A b

with 0.1 mM DTr, 0.2 M KCl

Elution: 3 volumes buffer A b with 0.1 mM DTr, 0.3 M KC1

Superdex 75 16/60

Buffer A, b 0.1 M KCI

a Anion-exchange cellulose DE52 is from Whatman (Clifton, NJ) and Sephacryl S-100 HR, phenyl- Sepharose CL-4B, and Superdex 75 are from Amersham Pharmacia Biotech (Piscataway, NJ).

b Buffer A: 50 mM Tris-Cl, pH 7.6.

24 PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES [2]

Fractions containing the R2 protein are pooled and concentrated in a Amicon (Danvers, MA) concentrator and by centrifugation on a Centricon-30 microcon- centrator, after addition of 10% (v/v) glycerol. The concentrated R2 protein is then aliquoted and stored at - 8 0 °.

Reconstitution of Iron-Radical Center

The recombinant A. thaliana R2 protein is purified in the apoprotein form. 13

The iron/radical sites are then reconstitued anaerobically by the addition of 8 equivalents of ferrous ammonium sulfate and 80 equivalents of ascorbate as a reductant. After a 5-rain incubation, the solution is exposed to air, and iron excess is removed on a Sephadex G-25 column.

Iron content is determined as described by Fish. 15 The tyrosyl radical content is determined by EPR spectroscopy, as described next.

S p e c t r o s c o p i c P r o p e r t i e s of T y r o s y l R a d i c a l

The light absorption spectrum of ribonucleotide reductase from E. coli dis- plays several bands in the 300- to 500-nm region (Fig. l). Two intense absorption bands at 325 (e = I0000 M - I cm - l ) and 370 nm (e = 8700 M - l cm - I ) are char- acteristic for the presence of the dinuclear oxygen-bridged iron center. They have contributions from oxo-to-iron charge transfer transitions. The tyrosyl radical is characterized by a very sharp peak at 410 nm (e = 6600 M - l cm-1 ) and a shoulder at 390 nm (e = 7200 M -I cm-l) . In the case of mouse and plant RNR, the peak appears at a slightly larger wavelength.

The presence of a stable tyrosyl radical in class I RNR can be easily concluded from its X-band EPR spectrum at liquid helium temperature, which shows a char- acteristic doublet centered at g = 2.0. The experimental conditions for recording the EPR spectrum of the various radicals shown in Fig. 2 are indicated in the corresponding legend. As discussed later, microwave power saturation studies of the signals are required in order to record spectra under nonsaturating condi- tions, as the various radicals have very different behavior with regard to power saturation.

In the case of the E. coli enzyme, the large hyperfine interaction (about 20 G) that dominates the observed doublet signal at 9 GHz (Fig. 2a) originates from one of the two/%methylene hydrogens, which is oriented with a dihedral angle of 33 ° with respect to the Pz orbital on the adjacent carbon of the aromatic ring. The second hydrogen is instead situated in the plane of the ring, thus perpendicular to Pz and the hyperfine coupling to that hydrogen is negligible. Smaller hyperfine interactions are associated with the protons in the ortho positions of the aromatic

15 W. W. Fish, Methods Enzymol. 158, 357 (1988).

[2] TYROSYL RADICALS AND R N R s 25

(M.emj

0

Wavelength (urn)

FIG. I. Light absorption spectrum of E. coli protein R2.

s×l -

ring with respect to the carbon carrying the oxygen atom. ENDOR spectroscopy has been used to determine the spin density distribution and demonstrated that the radical is uncharged and not hydrogen bonded to donors in its environment within the protein. 16,17

Enzymes from different species are highly homologous. However, they give rise to three distinct EPR signals defined by the conformation of the methylene group of their tyrosyl radicals. In the case of enzymes from mouse andA. thaliana, for example, the dihedral angles are 50 and 70 ° rather than 30 and 90 °, and hyper- fine interaction thus occurs with both fl protons (1.8 and 0.7 mT) (Fig. 2b). A third type of signal is found in the case of the enzyme from M. tuberculosis or Salmonella typhimurium, indicating a different dihedral angle (Fig. 2c).

These radicals display a great variety of temperature dependence and mi- crowave power saturation properties as a consequence of variable distances from the iron center and thus of variable magnetic interactions with that center. For example, in the case of M. tuberculosis, the radical is easily saturable, indicating a long distance from the center; consequently, care should be taken to work at very low applied microwave power. 13

16 C. J. Bender, M. Sahlin, G. T. Babcock, B. A. Barry, T. K. Chandrashekar, S. E Salowe, J. Stubbe, B. LindstrSm, L. Petersson, A. Ehrenberg, and B.-M. Sjtiberg, J. Am. Chem. Soc. 111, 8076 (1989).

17 C. W. Hoganson, M. Sahlin, B.-M. Sjtiberg, and G. T. Babcock, J. Am. Chem. Soc. 118, 4672 (t996).

26 PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES [9.]

R - - O °

H X . . _ . . _ /

g = 2.0047

i , .

I mT

FIG. 2. X-band EPR spectra of the tyrosyl radical from various protein R2: (a) E. coli (temperature, 10K; microwave power, 0.2 #W; microwave frequency, 9.47 GHz; modulation amplitude, 1.4 G); (b) A. thaliana (temperature, 26K; microwave power, 0.1 mW; microwave frequency, 9.2 GHz; mod- ulation amplitude, 1.0 G); and (c) M. tuberculosis (temperature, 30K; microwave power, 10 /zW; microwave frequency, 9.62 GHz; modulation amplitude, 1.92 G).

High-field (245-286 GHz) EPR spectroscopy at a low temperature is also a useful method to measure the g values of the tyrosyl radicals with high accuracy.12' 13,18-20 High-field EPR spectra (286 GHz) were recorded at 4K using the high-field EPR spectrometer at the Grenoble High Magnetic Field Laboratory (Fig. 3). The absolute accuracy of the g values is about 4-2 x 10 -4. Al l spectra

[2] TYROSYL RADICALS AND RNRs 27

> m i , .

o

0 ..o w

O. I.U

2.012

i , , I , , , I , i i I , . . I , , . I ~ , ~

2.01 2.008 2.006 2.004 2.002 2

FIG. 3. High-field EPR spectra of R2 proteins: (a) E. coli R2 and (b) A. thaliana R2. EPR record- ing parameters are temperature, 4K; microwave frequency, 286 GHz; and modulation amplitude, 10G.

were collected under nonsaturating conditions and simulated using a g tensor- only model, and no hyperfine interactions were explicitly included. It appears that whereas the values for gy and gz are almost identical, in the range of 2.0045 and 2.0022, respectively, those for gx vary from one radical to another and can be di- vided in two classes (Fig. 3). The lower values (gx = 2.0075-2.0078) ofgx found in the case of mouse and A. thaliana enzymes have been interpreted as reflect- ing the stabilization of the nonbonding orbitals of the oxygen through hydrogen- bonding interactions with proton donors in the protein. 13'2° Instead, the large values (gx = 2.0089-2.0092) found in the enzymes from E. coli, S. typhimu-

rium, or M. tuberculosis indicate that the tyrosyl radical is not hydrogen bonded.12,18,19

18 S. Un, M. Atta, M. Fontecave, and W. Rutherford, J. Am. Chem. Soc. 117, 10713 (1995). 19 p. Allard, A. L Barra, K. K. Andersson, P. P. Schmidt, M. Atta, and A. Gr~lund, J. Am. Chem. Soc.

118, 895 (1996). 2o p. p. Schmidt, K. K. Andersson, A. L. Barra, L. Thelander, and A. Gr'~lund, J. Biol. Chem. 271,

23615 (1996).

28 PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES [2]

R e a c t i v i t y o f T y r o s y l R a d i c a l

The radical nature of ribonucleotide reductase and, consequently, the involve- ment of key free radical intermediates, either protein or substrate bound, during catalysis are the specific characteristics of this family of enzymes. Still relatively few enzymes share this property. It then makes these radicals attractive targets for a selective inhibition of ribonucleotide reductase, and radical scavengers proved to be efficient inhibitors of DNA synthesis.

Assay for Tyrosyl Radical Scavenging Using EPR Spectroscopy

The R2 protein is diluted in 150/zl 0.1 M Tris-C1, pH 7.5, in order to have a final concentration of tyrosyl radical of 10 /zM. A control EPR spectrum of this solution is recorded. Calibration can be carried out using a TEMPO (2,2,6,6- tetramethyl- 1-piperidinyloxy, free radical) standard spectrum recorded under iden- tical conditions. After addition of a small volume of the radical scavenger solution directly in the EPR tube, incubation is carded out at 37 ° and stopped after 10 min by freezing the tube in a liquid N2 bath. A spectrum is recorded, and the relative amplitude of the characteristic tyrosyl radical signal at g = 2.00 (normalized to 100% for the control) is used to measure the inactivation of R2. Classically, the radical scavenger concentration used varies from 0.1 to 1 mM.

Figure 4 illustrates the results found with two radical scavengers, resveratrol (3,4',5-trihydroxy-trans-stilbene) and paracetamol (4-acetamidophenol), and the greater resistance of prokaryotic tyrosyl radicals, such as those from E. coli and M. tuberculosis, as compared to eukaryotic ones. The reactivity of the R2 protein from mouse is comparable to that of R2 protein from A. thaliana. 12

Radical Scavengers

Hydroxamic acids and polyphenols are efficient scavengers of RNR tyrosyl radicals.21-23 Hydroxyurea, for example, is used in clinics as an anticancer agent. It has been found to potentiate the anti-HIV activity of nucleoside analogs such as AZT and ddI. 24'25 Unfortunately, hydroxyurea-dependent inhibition is reversible, as cells contain activities that efficiently reincorporate the tyrosyl radical into protein R2. 26,27 Furthermore, cells rapidly develop resistances to the drug. High

21 I. Kj611er-Larsen, B.-M. Sj/Sberg, and L. Thelander, Eur. Z Biochem. 125, 875 (1982). 22 H. L. Elford, G. Wampler, and B. Van't Riet, CancerRes. 39, 844 (1979). 23 M. Atta, N. Lamarche, J. P. Battioni, B. Massie, Y. Langelier, D. Mansuy, and M. Fontecave,

Biochem. J. 290, 807 (1993). 24 S. D. Malley, J. M. Grange, E Hamedi-Sangsari, and J. R. Vila, Proc. Natl. Acad. Sci. U.S,A. 92,

11017 (1994). 25 W.-Y. Gao, A. Cara, R. C. Gallo, and F. Loft, Proc. Natl. Acad. Sci. U.S.A. 911, 8925 (1993). 26 M. Fontecave, R. Eliasson, and P. Reichard, J. Biol. Chem. 262, 12325 (1987). 27 M. Fontecave, R. Eliasson, and P. Reichard, J. Biol. Chem. 264, 9164 (1989).

[2] TYROSYL RADICALS AND R N R s 29

100

8O

60

e,i 40

20

~ E. coli ] M. tuberculosis A. t.l~_!i_a.na

Resveratrol

1 mM

Paracetamol

FIG. 4. Compared reactivities of tyrosyl radicals from E. coli, A. thaliana, and M. tuberculosis. Assays were carried out as described in the text using resveratrol or paracetamol as the radical scavenger. Percentage R2 activity represents the remaining EPR amplitude of the radical signal, which varies from 100 (no scavenging) to 0 (total scavenging). Recording conditions: temperature, 100K; modulation amplitude, 3.12 G; and microwave power, 10 (R2ec, R2at) or 0.3 (R2mt) mW.

doses, which eventually become toxic, are thus required. There is still a need for less toxic and more efficient inhibitors of ribonucleotide reductase. We have found that resveratrol, a natural phytoalexin found in grapes, is such an inhibitor and might be an attractive compound to investigate as an anticancer agent in humans. 28

Nitric Oxide (NO) and Superoxide Radical

Among a number of biological functions, NO functions as a mediator of the cytotoxic effects of activated macrophages. 29 It is responsible for the profound inhibition of DNA synthesis in target tumor cells, bacteria, intracellular parasites, or viruses, thus contributing to the host immune defense against rapidly prolifer- ating pathogens. Ample evidence now shows that ribonucleotide reductase is one of the key targets of NO. 3° During reaction, both proteins R1 and R2 are affected: the first one through nitrosylation of essential cysteines and the other through a coupling reaction of NO to the tyrosyl radical of R2. 31

Finally, RNR is an enzyme rather sensitive to oxidative stress conditions and needs to be protected by superoxide dismutase and catalase. 32 This is likely to be

28 M. Fontecave, M. Lepoivre, E. Elleingand, C. Gerez, and O. Guittet, FEBS Lett. 421, 277 (1998). 29 p. L. Feldman, O. W. Griffith, and D. J. Stuehr, Chem. Eng. News 20 December 26, (1993). 30 M. Lepoivre, J. M. Flamand, and Y. Henry, J. Biol. Chem. 267, 22994 (1992). 31 B. Roy, M. Lepoivre, Y. Henry, and M. Fontecave, Biochemistry 34, 5411 (1995). 32 M. Fontecave, A. Gr'~lund, and E Reichard, J. Biol. Chem. 262, 12332 (1987).

30 PROTEIN SENSORS AND REACTIVE OXYGEN SPECIES [3]

the consequence of the efficient and irreversible coupling reaction of superoxide radical to the enzyme tyrosyl radical. 33 This is an interesting observation because there are exceedingly few examples to date of direct reactions of superoxide with a biological target that would explain its toxicity and a need for superoxide dis- mutase in aerobic organisms. That ribonucleotide reductase might be a target for superoxide radicals is a notion that has been very little considered, so far.

33 p. Gaudu, V. Nivi~re, Y. Prtillot, B. Kauppi, and M. Fontecave, FEBS Lett. 387, 137 (1996).

[3] Flavin-Dependent Sulfhydryl Oxidases in Protein Disulfide Bond Format ion

By KAREN L. HOOBER a n d COLIN THORPE

I n t r o d u c t i o n

Both metal- and FAD-dependent sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with a concomitant reduction of molecular oxygen to hydrogen peroxide.

2 R-SH + 02 ~ R-S-S-R + H202 (1)

Despite long-standing suggestions that these enzymes contribute to disulfide bond formation in eukaryotes, 1-6 they have been widely and surprisingly neglected. An earlier volume in this series presented procedures for both iron- and copper- dependent sulfhydryl oxidases. 7-9 The present contribution addresses flavin-linked enzymes.

There are two evolutionarily unrelated classes of flavin-linked sulfhydryl ox- idases. That secreted from Aspergillus niger contains FAD and a redox-active cystine bridge 4 and is most closely related to alkyl hydroperoxide reductase in

I V. G. Janolino and H. E. Swaisgood, J. Biol. Chem. 250, 2532 (1975). 2 H. E. Swaisgood and H. R. Horton, Ciba Found. Symp. 72, 205 (1980). 3 M. C. Ostrowski and W. S. Kistler, Biochemistry 19, 2639 (1980). 4 R. S. de la Motte and E W. Wagner, Biochemistry 26, 7363 (1987). 5 D. A. Clare, I. B. Pinnix, J. G. Lecce, and H. R. Horton, Arch. Biochem. Biophys. 265, 351 (1988). 6 K. Takamori, J. M. Tholpe, and L. A. Goldsmith, Biochim. Biophys. Acta 615, 309 (1980). 7 M. X. Sliwkowski and H. E. Swaisgood, Methods Enzymol. 143, 119 (1987). 8 H. E. Swaisgood and H. R. Horton, Methods Enzymol. 143, 504 (1987). 9 L. m. Goldsmith, Methods Enzymol. 143, 510 (1987).

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METHODS IN ENZYMOLOGY, VOL. 348 0076-6879102 $35.00