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nl/locate/jinorgbio Journal of Inorganic Biochemistry 78 (2000) 149160

Hydrogen abstraction from thiols by adenosyl radicals: chemical precedent for thiyl radical formation, the rst catalytic step in ribonucleoside triphosphate reductase from Lactobacillus leichmannii
Jeanne M. Sirovatka, Richard G. Finke *
Chemistry Department, Colorado State University, Ft. Collins, CO 80523, USA Received 7 July 1999; received in revised form 2 November 1999; accepted 23 November 1999

Abstract Aqueous solutions of adenosylcobalamin (AdoCbl) were thermolyzed with excess b-mercaptoethanol under anaerobic conditions. The product studies reveal that ;90% CoC bond homolysis occurs, to yield Co(II)cobalamin, 59-deoxyadenosine, and the disulde product from the combination of two HOCH2CH2Sx radicals, 2,29-dithiodiethanol; there is also ;10% CoC bond heterolysis, yielding Co(III)cobalamin, adenine, and 2,3-dihydroxy-4-pentenal. The kinetic studies show there is a rst-order dependence on AdoCbl and zeroorder dependence on thiol under the higher [RSH] experimental conditions used, consistent with the rate-determining step at high [RSH] being the generation of Adox. The kinetic results require that, in enzyme-free AdoCbl solution, adenosyl radical (Adox) is formed as a discrete intermediate which then abstracts Hx from the added thiol. The activation parameters for CoC bond homolysis in the presence of thiol trap are the same within experimental error as the activation parameters for CoC bond homolysis without trap, DHobss29(2) kcal moly1 and DSobssy1(5) e.u. The results, in comparison to the rate of CoC bond homolysis in ribonucleoside triphosphate reductase (RTPR), reveal that RTPR accelerates CoC bond cleavage in AdoCbl by ;1010"1. The recent literature evidence bearing on the exact mechanism of RTPR enzymic cleavage of the CoC bond of AdoCbl is briey discussed, notably the fact that this mechanism is presently controversial, but does involve at least coupled (and possibly concerted) CoC cleavage, SH cleavage, and CH (AdoH) formation steps. q2000 Elsevier Science Inc. All rights reserved.
Keywords: Hydrogen abstraction; Thiols; Adenosyl radicals; Ribonucleoside triphosphate reductase; Coenzyme B12; Adenosylcobalamin

1. Introduction

Ribonucleotide reductases catalyze the transformation of ribonucleotides to deoxyribonucleotides in the rate-determining step of DNA biosynthesis [1]; hence, all living cells require a ribonucleotide reductase [2,3]. There are currently four classes of ribonucleotide reductases, determined by their enzymatic cofactor [4]: Class I contains a diferric cluster and an observable tyrosyl radical; Class II contains coenzyme B12 (adenosylcobalamin; AdoCbl, Fig. 1), and is the class of interest to the present work; Class III contains a FeS cluster and an observable glycyl radical; and the less studied Class IV enzymes, which are postulated to contain a dimanganese cluster and a tyrosyl radical. Relevant to the present work is the fact that all of the ribonucleotide reductases are proposed
* Corresponding author. Fax: q1-970-491-1801; e-mail: rnke@ lamar.colostate.edu Fig. 1. Coenzyme B12, also known as adenosylcobalamin (AdoCbl), and the schematic representation of AdoCbl used in the schemes herein.

0162-0134/00/$ - see front matter q2000 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 2 2 4 - X

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Fig. 2. A schematic of the proposed active site of RTPR, based on the active site sequence homology between RTPR and RDPR, the X-ray crystal structure of RDPR, and EPR studies of Stubbe et al. indicating a Cox---xSR radical pair as discussed and referenced in the main text.

to involve a protein-based cysteinyl (thiyl, RSx) radical mechanism [4] 1. Coenzyme B12-dependent ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii, a Class II enzyme [57], is of special interest due to the following four reasons: (i) it is one of the smallest ribonucleotide reductases, with a molecular weight of 81.8 kDa [8]; (ii) it has been cloned and overexpressed [8]; (iii) an effective afnity column for the purication of over-expressed RTPR to crystallographic-level purity is now available [9,10], so that highly puried protein for biochemical studies is now available; and, perhaps most importantly, (iv) denitive evidence has been reported by Stubbe and van der Donk for cysteinyl radical involvement in the catalytic cycle (vide infra) [4]. Coenzyme B12-dependent RTPR is, therefore, an ideal system
1 There is also evidence that other enzymes, such as pyruvate formatelyase, utilize a protein-based cysteinyl radical in their catalytic cycles [4].

on which to focus both chemical precedent studies and enzyme mechanistic studies. The literatures proposed active site for RTPR is shown schematically in Fig. 2 [11]. The proposed active site is based on the signicant sequence homology of RTPR with the crystallographically characterized [1214] Class I ribonucleotide reductase, ribonucleoside diphosphate reductase (RDPR) from Escherichia coli [15,16]. The active site sequence homology between RTPR and RDPR, along with the similarity in catalytic function and inhibition mechanisms between the two enzymes [4], supports the proposed RDPRlike active site for RTPR (Fig. 2) [11]. The postulated presence of an asparagine residue in RTPRs active site is supported by the presence of the N437 residue in an analogous position in the crystal structure of the active site of RDPR [13], but remains to be experimentally veried or refuted. The three cysteine residues, C408, C419, and C119 in Fig. 2, have been shown to be required for catalytic activity in RTPR [17] (and also RDPR [16]). Specically, cysteine C408 has been shown to be absolutely required for catalysis since mutating C408 to a serine completely eliminates all catalytic activity in the enzyme [17]. In addition, Stubbe and co-workers [18,19] have provided EPR evidence that RTPR contains a coupled Co(II)x---xS-C408 radical pair in the active This seminal work by the Stubbe site, separated by 57 A. group provides the rst direct evidence of a protein-based, cysteinyl radical at the active site of any enzyme. The proposed, minimum mechanism for RTPR that one nds in the literature [11] is shown in Fig. 3. The catalytic cycle is initiated by homolytic CoC bond cleavage of AdoCbl, which results, at least formally, in the formation of

Fig. 3. A minimal proposed mechanism for RTPR based on the literature. The focus of the present work is on the AdoCbl CoC cleavage in the presence of thiols and resultant formation of a thiyl radical, RSx, and AdoH.

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Co(II)cobalamin and a putative, carbon-centered 59-deoxyadenosyl radical, Adox. However, direct evidence for the Adox radical has never been obtained in RTPR or, for that matter, in any other B12-dependent enzymatic system. The rst observable products of CoC bond homolysis, with RTPR and at the active site, are Co(II)cobalamin and 59-deoxyadenosine (AdoH) plus the C408-based thiyl radical, C408Sx [4,2027]. Stubbe and co-workers [28,29] have proposed that Hx abstraction from C408 is concerted with CoC bond cleavage, although transient formation of an Adox intermediate could not be unequivocally ruled out [30]. The resultant thiyl radical, C408-Sx, then abstracts Hx from the substrate, which is followed by substrate radical b-OH loss, and eventual product formation (Fig. 3). A paper by Lenz and Giese [31] provides chemical precedent for b-OH elimination following generation of the substrate-based radical. Recent results by the Stubbe group [32] indicate that the CoC bond is reformed during each catalytic cycle, resulting in a radicalchain length of one in the enzymatic system, and indicate that, at least under conditions where [RTPR]4[AdoCbl], the AdoCbl cofactor can be passed from one RTPR molecule to another RTPR molecule. Although it is well established that alkyl free radicals abstract Hx from thiols at rates of 106108 My1 sy1 [3341], an exact chemical precedent for the AdoCbl CoC cleavage, RSH cleavage, and CH (AdoH) bond formation step(s) has not yet been provided [42,43] 2,3. In particular, studies are needed to provide chemical precedent for or against the proposed stepwise [4450] 4 or concerted reaction of AdoCbl with a thiol. These two possible mechanisms are shown in Scheme 1. Herein, we report a detailed chemical precedent product, kinetic, and mechanistic studies of the reaction of adenosylTwo reports generating adenosyl radicals in the presence of thiols have been published: one by Rudakova et al. [42], and one by Hogenkamp et al. [43]. Rudakova et al. [42] reported acetaldehyde formation from the photolytic reaction of AdoCbl with the dithiol dihydrolipoic acid amide and ethylene glycol in pH 2.0 aqueous buffer (presumably at room temperature; the time of reaction was also not reported). However, no direct evidence was given for the generation of thiyl radicals (e.g., by the putative reaction of AdoxqRSH), nor for the participation of thiyl radicals, RSx, in acetaldehyde formation; also unavailable from the preliminary communication is a complete, balanced reaction stoichiometry. Hogenkamp et al. [43] reported that adenosylthioether, AdoS CH2CH2OH, is formed from the thermolysis of AdoCbl with b-mercaptoethanol at 70 8C over 3 days. The formation of the thioether product, Ado SCH2CH2OH, can be rationalized by Adox abstracting a Hx from b-mercaptoethanol, and then the resultant xSCH2CH2OH combining with a second Adox to form AdoSCH2CH2OH. However, the full stoichiometry of the reaction was again not reported, nor is any kinetic or mechanistic analysis available that would have allowed one to answer the specic questions raised in the present paper (questions (i)(iii), main text). 3 We note that the reverse of the AdoCblqRSHAdoHqRSxqxCbl reaction, namely, the re-formation of AdoCbl from AdoH plus RSx and x Cbl, is of greater mechanistic interest. However, the transition state for both reactions is the same by the principle of microscopic reversibility. 4 The stepwise mechanism of Rx formation, followed by Hx abstraction from R9SH by Rx, has been demonstrated by Halpern and co-workers [50] for thermolysis of the B12-model complex [py(saloph)CoCH(CH3)2] in the presence of n-C8H17SH.
2

cobalamin with b-mercaptoethanol with the goal of answering the following three questions. (i) Is Hx abstraction from thiol by an adenosyl radical concerted or stepwise in solution? (ii) If concerted, is there a substantial rate acceleration of CoC homolysis due to a little-precedented intermolecular molecule-assisted homolysis [5154]? (iii) Is the enzymic acceleration of CoC cleavage by RTPR the same as the 1012"1 rst documented elsewhere for other B12-dependent enzymes using the non-thiol trap, TEMPO [49]? Until now, no study of AdoCblqRSHAdoHqRSxqCo(II)Cbl has been completed, and hence no direct comparison has appeared of the rate laws, mechanism, or absolute rates of CoC cleavage of AdoCbl plus thiol thermolysis in solution versus the rate of enzymic CoC cleavage by RTPR.

2. Results and discussion 2.1. AdoCbl plus thiol CoC bond thermolysis product studies In an initial control experiment, thermolysis of an aqueous solution of ;1=10y4 M AdoCbl with no thiol yields the same products reported earlier for aqueous AdoCbl thermolysis without trap [55]: 100(5)% Co(II)Cbl, ;75% cyclicAdo (the self-trapped, Adox homolysis product), ;5% 59-deoxyAdo (the homolysis product trapped by an exogenous Hx); and ;10% adenine (from CoC heterolysis) (Table 1, see also Scheme 1). This control establishes that AdoCbl thermolyses provide reproducible results between different investigators in our laboratories and in the absence of thiol. The fact that three other labs have quantitatively reproduced our earlier AdoCbl plus TEMPO results [44] is reassuring as well [5658]. Thermolysis of aqueous solutions of ;1=10y4 M AdoCbl in the presence of excess b-mercaptoethanol (0.02 0.1 M), Table 1 results in ;90% 59-deoxyAdo and, hence, ;90% homolytic cleavage of the CoC bond, plus ;10% adenine, signifying ;10% of a CoC heterolysis pathway [55] (Scheme 2). No detectable (F3%) cyclic-Ado is present when G0.02 M thiol is added. The dramatic change in the product distribution with G0.02 M b-mercaptoethanol as illustrated in Table 1 is readily explained by the previously established [44] mechanism (Scheme 1), in which an Adox intermediate is formed, then efciently trapped by added RSH serving as an exogenous Hx source, ktrap,thiol 5. The sole nal cobalamin product (100"5%) from the combined 90% homolysis and 10% heterolysis reaction is Co(II)Cbl. Note that although the ;10% of CoC heterolysis must give Co(III)Cbl initially, this species is well known to be rapidly reduced by the aldehyde, 2,3-dihydroxy-4-pen5 The amount of heterolysis is not dependent on [RSH], but is a function of solvent: ;10% heterolysis is seen in water, both in the absence [55] and presence of thiol, while no detectable heterolysis (F3%) is seen in ethylene glycol, both in the absence [44] and presence of (0.1 M) thiol.

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Scheme 1. Precedented (top) and possible (middle and bottom) mechanisms for the formation of AdoCbl homolysis products in the presence and absence of radical traps. The mechanism of the net Hx loss from the cyclic-Adox radical is not known with certainty, but probably involves Hx abstraction by the persistent Co(II)Cbl radical [80], then precedented disproportionation of the resultant HCo(III)Cbl, 2HCo(III)Cbl|H2q2Co(II)Cbl as discussed elsewhere [44,81].

tenal (produced by the heterolytic CoC cleavage, Scheme 2), to form Co(II)Cbl and an oxidized organic product [44]. 2,29-Dithiodiethanol, the expected disulde product from the diffusion-controlled combination of two HOCH2CH2Sx thiyl radicals [59], was detected and quantitated by GLC using DMF as an internal standard. The amount of disulde detected was the expected, stoichiometric 0.5 equiv. of RS SR (Scheme 1) within "10% experimental error (Table 1). Moreover, no other thiol-derived product (e.g., no AdoSR [43]) could be detected. The quantitative yield of disulde provides strong evidence for thiyl radical formation, x SCH2CH2OH (Scheme 1). Note, however, that the products alone do not distinguish between a concerted or a stepwise mechanism, the focus of question (i) listed in Section 1. The kinetic studies discussed next address question (i). 2.2. AdoCbl plus thiol CoC bond thermolysis kinetic studies The thermolysis kinetics of AdoCbl with 0.020.1 M bmercaptoethanol, utilizing the exact same solutions used for

the product studies, were followed by UVVis spectroscopy (Fig. 4) and analyzed according to ln[A`/(A`yAt)] versus time plots (Fig. 5). The results in Table 1 show that the observed rate of CoC bond thermolysis is independent of [RSH] above 0.02 M. Hence, the reaction is zero-order in [RSH] over the range studied. (In addition, kobs at high [RSH] (1.0 M) was measured to be kobss14(3)=10y5 sy1, within experimental error of kobs at low [RSH], demonstrating that the reaction is zero-order in thiol even at very high [RSH].) This fact, plus the fact that the observed products (i.e., HOCH2CH2SSCH2CH2OH) are derived from the added RSH, demands that the reaction mechanism consists of at least two steps. Hence, we have our answer to question (i): the mechanism of AdoCbl CoC cleavage in solution and in the presence of RSH is stepwise, not concerted. Question (ii) is also, therefore, answered: in solution there is no acceleration of CoC cleavage due to a concerted, moleculeassisted homolysis. In addition, the kobs obtained for CoC thermolysis in the presence of excess thiol, kobss10(2)=10y5 sy1, is the same

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J.M. Sirovatka, R.G. Finke / Journal of Inorganic Biochemistry 78 (2000) 149160 Table 1 Nucleoside product and kinetic values for the thermolysis of AdoCbl with b-mercaptoethanol a [Thiol] % Adenine b % 59-DeoxyAdo % Cyclic-Ado % Total nucleoside mass balance 92 97 99 94 % RSSR

153

kobs=105 (sy1)

0 0.02 0.05 0.1

a b

12(3) 8(3) 7(3) 13(3)

5(3) 89 92 81

75 0 0 0

0 106(10) 98(10) 108(10)

6.8(2) 9.2(3) 11(2) 9.9(3)

Error bars on the nucleoside products are "5% unless indicated otherwise. Note these adenine yields are within experimental error of their average of 9"3%, and thus are not a function of the RSH concentration.

Scheme 2. The AdoCbl plus thiol homolysis plus heterolysis reaction and resultant product stoichiometry.

within experimental error as the kobs obtained for CoC thermolysis in the presence of excess TEMPO in ethylene glycol, kobss12.1(1)=10y5 sy1 [60]. This nding provides further evidence for the stepwise trapping mechanism shown in Scheme 1, and further reveals that the change in solvent from ethylene glycol to water does not signicantly affect kobs as expected for a radical (homolysis) mechanism. Hence, the product and kinetic data for CoC bond thermolysis of AdoCbl are completely consistent with, and indeed highly supportive of, reversible CoC homolysis (at low [RSH]),

followed by the radical-trapping mechanism shown in Scheme 1 6. 2.3. Activation parameters Solutions of ;1=10y4 M AdoCbl in the presence of 0.1 M b-mercaptoethanol (i.e., under zero-order [RSH] condi6 Note that the kobs value without thiol is non-zero; this is due to the known self-trapping reaction of Adox by cyclization [44] to form cyclic-Ado, as was seen in the control experiment for thermolysis of AdoCbl alone [49].

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Fig. 4. Visible kinetic trace of ;1=10y4 M AdoCbl and 0.1 M b-mercaptoethanol at 100 8C over 24 h in pH 7.0 buffered water. Isosbestic points are seen at 390, 480 and 575 nm. The absorbance increase at 474 nm was used to obtain the kinetic data.

Fig. 5. Plot of ln[A`/(A`yAt)] vs. time (at 474 nm) of the thermolysis of ;1=10y4 M AdoCbl and 0.1 M b-mercaptoethanol at 100 8C over 24 h (12 half-lives) in pH 7.0 buffered water. The rst 7 h (4 half-lives) are shown.

tions) were thermolyzed in a temperature range of 85110 8C in order to determine the activation parameters of the Co C bond cleavage step. A plot of ln[kobs/T] versus 1/T over a 25 8C temperature range (Fig. 6) is linear within experimental error, and gives values of DHobss29(2) kcal moly1

and DSobssy1(5) e.u. These values are in good agreement with those previously reported for the thermolysis of AdoCbl in the absence of trap in aqueous solution, DHobss30.9(4) kcal moly1 and DSobss4.8(8) e.u. [55], as well as those obtained using the non-thiol trap, TEMPO (in ethylene glycol), DHhs30.6(3) kcal moly1 and DShs2.9(7) e.u. [44]. The fact that the values obtained herein with RSH trap are equivalent to the values obtained without trap conrms that trap is not involved in the rate-determining step of AdoCbl CoC bond cleavage in aqueous solution. As detailed elsewhere, the actual rate-determining step is diffusion of Adox out of the solvent cage [46]. Comparing the solution CoC cleavage rate constant extrapolated 7 from our activation parameters in the 85110 8C range to 37 8C, kobs(37 8C)f10y9 sy1, with the specic forward rate constant of Co(II)Cbl formation in RTPR, derived from the Keq value for reversible Co(II)Cbl formation in the enzyme, kfs1455 sy1 at 37 8C [29,30], reveals an enzymatic acceleration of 1010"1 for CoC bond cleavage in RTPR over our solution, intermolecular RSH plus AdoCbl system. This, then, provides the answer to question (iii): the enzymic acceleration measured for B12-dependent RTPR is 1010"1, a value within experimental error to what we previously determined for diol dehydratase, 1012"1 [49]. Note also that kobs, and thus DHobs and DSobs, are composites containing well-established DH8 and DS8 contributions from the temperature dependence of the base-on, base-off Keq [55]. Using the previously-determined DH and DS values for the base-on, base-off Keq in water [55], one can estimate the values for base-on AdoCbl thermolysis in the presence of thiol to be DHh(base-on)s30"2 kcal moly1 and DSh(base-on)s4"6 e.u., respectively (see Section 4 for the needed derivation), values that are the same within experimental error (i.e., as they had to be since DHobs and DSobs were the same within experimental error) as those obtained previously in aqueous solution, but without thiol present [55], DHh(base-on)s33"2 kcal moly1, DSh(base-on)s11"3 e.u. 2.4. AdoCbl plus cysteine thermolysis studies In addition to studies with b-mercaptoethanol, reactions using the biologically more relevant thiol, cysteine, were investigated. The nucleoside products for ;1=10y4 M AdoCbl with 0.1 M cysteine present were the same within experimental error as those for b-mercaptoethanol: ;95% 59-deoxyAdo and ;9% adenine were detected. However, the use of cysteine concentrations less than 0.1 M result in incomplete mass balance in the detected nucleoside products (0.01
7 That is, this comparison makes the necessary assumption, in the absence of a direct measurement of the very slow AdoCbl CoC cleavage at 37 8C [57,61], that this extrapolation does not cause more than, say, about 1012 error bars over the range of the extrapolation of about 50 8C. Note also that even an error of 1012 (or more) in no way inuences the primary conclusion [62] that B12 enzyme causes an enormous acceleration of CoC bond cleavage.

Fig. 6. Plot of ln[kobs/T] vs. 1/T for the thermolysis of ;1=10y4 M AdoCbl with 0.1 M b-mercaptoethanol in pH 7.0 buffered water from 85 to 110 8C.

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M cysteine gave 8% adenine, 5% cyclic-Ado, and 61% 59deoxyAdo; hence, 26% of the nucleoside products are missing under these lower [cysteine] conditions; see also Fig. E, Section 4, which shows a continuous decrease in the total mass balance at [cysteine]-0.1 M). This incomplete mass balance at lower [cysteine] is likely due to the formation of adenosyl-thioether products, for example the AdoS CH2CH2OH product that Hogenkamp et al. [43] isolated when reacting AdoCbl with 20 equiv. of b-mercaptoethanol. However, we did not investigate the [cysteine]-0.1 M reactions further, since high [cysteine] conditions provide complete nucleoside stoichiometry, as discussed next. For [cysteine]G0.1 M, a white precipitate formed in the reaction vessel, which we have unequivocally identied as the disulde, cystine, based on its decomposition temperature and by ESMS in comparison to authentic, commercial product. In addition, H2S(g) was qualitatively detected by its precipitation as PbS(s) following the addition of Pb(OAc)2. Incomplete formation of cob(II)alamin in this AdoCbl plus G0.1 M cysteine thermolysis is also seen, a loss of isosbestics becoming detectable after 7 h of reaction (Fig. 7). The loss of isosbestics in the reaction and observed products can be explained by the reactions in a precedented [62,63] radicalchain mechanism (Scheme 3). This scheme predicts the formation of HSx, H2S, and HSCo(III)Cbl plus Co(II)Cbl; the cobalamin spectrum that develops (Fig. 7(b), 7 h spectrum lmaxs370, 410, and 530 nm; 20 h spectrum lmaxs370 (sh), 410 (sh), and 530 nm) is quite similar to that reported for hydrosuldocobalamin (lmaxs270, 350, 415, and 528 nm) [64]. (The other possible cobalamin product, cysteinylcobalamin, has been omitted from this scheme since we have prepared it independently and shown it to be metastable under aqueous, pH 7, conditions (t1/2-1 min at pH 7.2, 15 8C) [65].) The kinetics of the AdoCbl plus cysteine reaction were analyzed by a ln[[AdoCbl]i/[AdoCbl]t] versus time plot throughout the ;7 h time during which the isosbestics are maintained. Observed rate constants for the reaction of AdoCbl with cysteine are again independent of cysteine concentration; kobs(100 8C)s7.7(3)=10y5 sy1 for concentrations of cysteine ranging from 0.002 to 1.0 M. This observed rate constant is the same as that seen with b-mercaptoethanol (kobs(100 8C)s10(2)=10y5 sy1) within experimental error, indicating that the initial CoC cleavage (initiation) step in the presence of cysteine occurs by the same ratedetermining CoC cleavage (then diffusion out of the solvent cage) mechanism shown in Scheme 1. To summarize, the reactions shown in Scheme 3 provide a precedented [62] explanation 8 for the cysteine-derived
8 We attempted to check the thermochemical viability of the degradation of cysteine to H2S and qH3NC(CH2)COOy using Bensons heats and entropies of formation [66], but found that one cannot do this calculation due to the lack of data for CD-(N)(CD) and N-CD(H)2 terms, using Bensons nomenclature. If one simplies the system to CH3CH2CH2SH degrading to H2S and CH2_CHCH3, then one obtains a DG8 (100 8C)s3.73 kcal/mol by Bensons thermochemical additivity data. Such slightly thermodynami-

Fig. 7. Visible spectra of thermolysis of ;1=10y4 M AdoCbl (a) with 2=10y3 M cysteine and (b) with 0.1 M cysteine in pH 7.0 buffered water at 100 8C. Note the loss of isosbestics after 7 h in both (a) and (b) at 380 nm; the higher [cysteine] in (b) generates a much more dramatic deviation from the isosbestic point at 380 nm.

thiol products formed after the initial Hx abstraction from cysteine by Adox. The postulated mechanism in Scheme 3 is further fortied by (i) pulse radiolysis kinetic studies of Zhao and Lind [62] which provide indirect evidence for the carbon-centered radical derived from cysteine, and (ii) EPR studies of Grierson et al. [67] directly detecting the related, carbon-centered radical derived from glutathione. 2.5. Mechanistic and biological implications Two important results from this study are that the thermolysis of AdoCbl in the presence of thiol generates ;90% Adox, which then abstracts hydrogen from thiols quantitatively to yield 59-deoxyadenosine and a thiyl radical, RSx. The ;90% CoC homolysis efciency, with only ;10% CoC bond heterolysis, occurs even in solution and when the thiol is not held closely adjacent and intramolecularly, as it is at the active site of RTPR. Hence, the AdoCbl plus thiol intramolecular step in RTPR to yield AdoH and C408-Sx is predicted on the basis of these chemical precedent studies to be 490% (i.e., quantitatively) efcient at producing thiyl radicals. Our chemical precedent studies also imply that one role of the presumably hydrophobic active site of RTPR is to
cally uphill reactions would be driven by the loss of the volatile H2S(g) under the reaction conditions.

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Scheme 3. Precedented [62,64] radical-chain mechanism for the probable subsequent reactions of cysteinyl radical formed by the initial Hx abstraction from cysteine by Adox. The cross-termination steps, of cysteinex and HSx, or of xCo(II)Cbl and cysteinex, are also possible, but have been omitted for the sake of clarity, and since we know cysteinylCbl is only metastable at pHG5.5 [65].

overcome the F10% CoC heterolysis seen herein for thiols plus AdoCbl in protic, aqueous solution. (The axial base in AdoCbl may also be involved in enzymatic control of AdoCbl CoC homolysis over heterolysis; see elsewhere for further information on this point [6871].) This study provides, therefore, good chemical precedent for the quantitative stoichiometry of the proposed, AdoCblqprotein-SHAdo HqCo(II)Cblqprotein-Sx rst step in RTPRs postulated mechanism of action. A second important observation is that addition of thiol does not change the homolysis/heterolysis ratio of AdoCbl CoC bond thermolysis, nor does it change the activation parameters for the rate-determining step of CoC bond cleavage when excess thiol is present. In short, the rate law is zeroorder in thiol ([RSH]s0.021.0 M), requiring that thiols are not involved in the rate-determining step in AdoCbl Co C cleavage in solution, and do not affect the transition state of CoC bond cleavage; instead, thiol trapping of Adox occurs after a rate-determining CoC bond cleavage step and following Adox escape from the solvent cage [46]. These solution chemical precedent results stand in contrast to recent enzymic results interpreted as concerted CoC bond

cleavage, SH bond cleavage, and CH bond formation in RTPR [4,28]. One possible way to generate a concerted reaction in the enzymatic active site would be to enforce a very specic Co---C---H---S arrangement and bond distance, presumably one where the CHS angle would be near 1808 [30]. Such a transition state for CoC cleavage could result in molecule-assisted homolysis [51], and this new chemistry (i.e., new compared to the stepwise AdoCbl CoC cleavage in solution demonstrated herein) would be an important function of the enzyme. On the other hand, stepwise AdoCbl CoC bond cleavage in RTPR, via a transient [Adox], cannot be unequivocally excluded based on any of the available data [28]. Third, the estimated enzymic acceleration of AdoCbl Co C cleavage in RTPR is 1010"1. It remains to be claried 9 via
9 The mechanism and energetics of CoC cleavage in AdoCblxRTPRxdGTP are presently controversial. In two recent papers, the Brown [30]and Stubbe [28,29] groups have presented interesting, albeit conicting, evidence, data analysis, and interpretations. Browns group [30], using a composite mechanism for the AdoCblqRSHAdoHqRSxqCo(II)Cbl step, nds two different temperature regimes in the kinetic data, with a break point at 28 31 8C (outside the temperature region of Stubbes research, vide infra). At

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J.M. Sirovatka, R.G. Finke / Journal of Inorganic Biochemistry 78 (2000) 149160

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studies with RTPR whether this acceleration is through enzymic DH [30,48], DS [29], radical cage effects [48], corrinenzyme hydrogen-bonding interactions [72], corrin buttery conformational changes [73], or other (e.g., possible RTPR dimerization [10]) effects as discussed elsewhere [74]. Finally, the AdoCbl/b-mercaptoethanol chemical precedent system, and its now better understood mechanism of homolysis and subsequent Hx abstraction from a thiol, provides one system from which to study the second, even less precedented, step of the RTPR mechanism: abstraction of Hx from the substrate by a thiyl radical, RSx. Also of interest are the kH/kD values for Adox plus RSH/RSD, although we have shown (see Section 3, Experimental) that it is not possible to obtain this desired kH/kD by at least RSH/RSD competition experiments and under the [RSH] ([RSD]) and H2O (D2O) conditions utilized for the present study. Hence, efforts to obtain the Adox plus RSH/RSD kH/kD under the required different thiol and solvent conditions are in progress as part of what has already proved to require its own separate study.

thylformamide (Mallinckrodt) were used as received. bMercaptoethanol (Acros) was used as received, and stored under a layer of argon gas which had been passed through O2 and H2O traps. As a control to check the RSH purity, GC analysis of b-mercaptoethanol for disulde showed that F0.5% disulde contaminant is present in the b-mercaptoethanol. 3.2. Instrumentation UVVis spectra were recorded on an HP 8452A spectrophotometer equipped with a thermoelectric Peltier cell block temperature controller (25"1 8C). The spectrophotometer has a dynamic range of 0.0023.3 absorbance units at 350 nm and a manufacturers stated accuracy of "0.005 at 1.0 absorbance unit at 440 nm, so that it is possible to use it for absorbencies up to ;2.3 absorbance units. All kinetic samples were prepared under argon using a Schlenk line with Schlenk-type [55,60] UVVis cuvettes. The temperature of the oil bath (100.0"0.2 8C) employed in the kinetic experiments was calibrated using an NBS-calibrated thermometer with "0.2 8C gradations and controlled using a Barnant temperature controller. An HP 1050 HPLC (ls260 nm) detector equipped with an Alltech C18 Versapack reversed-phase column was used for nucleoside product analyses. Product quantitation was accomplished as before [70] using HPLC response factors generated from linear plots of peak area versus concentration of authentic product material. Thiol and disulde quantitation was accomplished using an HP 5890A Series II gas chromatograph with an HP 3395 integrator. The GLC was equipped with a Supelco b-dex 110 column, 30 m=0.25 mm. 3.3. AdoCbl plus RSH CoC thermolysis product studies To begin with, a control was performed to verify the reliability of the product data; specically, thermolysis of 1=10y4 M AdoCbl in H2O without thiol gave results equivalent within experimental error to work completed earlier by a different researcher in our previous labs at Oregon [55]: adenines12(3)% (versus 11(3)% previously), cyclicAdos78(5)% (versus 88(5)% previously) and 59deoxyAdos5(3)% (versus 1(3)% previously). A solution of 4 mL degassed pH 7.0 water (0.01 M sodium phosphate buffer), with 0.020.1 M b-mercaptoethanol and 5 mL DMF added, was degassed via three freezepumpthaw cycles and relled with argon. (Water was chosen as the solvent since it allows direct comparison with earlier AdoCbl thermolysis measurements [55].) In a darkened room lit only by red light, the solution was added using an airtight syringe to a Schlenk cuvette [55,60] containing ;0.7 mg AdoCbl under argon to form about a 1=10y4 M solution of AdoCbl. The Schlenk cuvette was then heated in the dark at 100.0"0.2 8C for ;24 h. The reaction was followed by UVVis spectroscopy at 474 and 522 nm until the reaction was judged complete (no change in the spectrum at 474 or 522 nm to 3

3. Experimental 3.1. Chemicals AdoCbl, obtained from Aldrich (G98% pure by HPLC), was stored below 0 8C, and used as received. DL-Cysteine and cystine were obtained from Aldrich, stored at 0 8C, and used as received. 2,29-Dithiodiethanol (Fluka) and dimetemperatures from 23 to 30 8C, Browns group reports activation parameters of DHfs42(4) kcal/mol and DSfs83(14) e.u., but at temperatures from 30 to 40 8C (the temperature range of Stubbes research and of enzymatic activity) Brown and co-workers report activation parameters of DHfs20(1) kcal/mol and DSfs13(4) e.u. From these data, Browns group concluded that, for temperatures above 30 8C, the activation of CoC bond cleavage is entirely enthalpic. They disfavor a concerted mechanism, arguing that any enthalpic stabilization of the concerted transition state, based on CH versus SH BDEs, could be at most only ;6 kcal/mol, insufcient to account for the 13 kcal/mol stabilization needed for the Co C bond cleavage acceleration. Contrasting this, in the rst of two papers of Stubbe and co-workers [28], evidence consisting of active site cysteine mutation and a global 20-parameter t to the isotopic kinetic data is interpreted in terms of a concerted Co---C bond cleavage, S---H bond cleavage, and C---H bond formation step. In the second paper by Stubbe and co-workers [29], the proposed concerted mechanism is then used to obtain activation parameters for the CoC bond cleavage step in the enzyme using a more limited temperature range of 33 40 8C: DH2s46(7) kcal/mol, DS2s96(12) e.u. From these data, Stubbe and co-workers conclude exactly opposite to Browns conclusion that the acceleration of the CoC bond cleavage is entirely entropic. A main experimental difference between the two studies is that Stubbes group runs their experiments under conditions of excess AdoCbl, while Browns group runs their experiments under conditions of excess RTPR. There are also obvious differences in the way the data are analyzed in the two studies, most notably Browns independent measurement of the Keq for AdoCbl binding to RTPR as a function of temperature, then deconvolution of the kobs for Co(II)Cbl formation using this independently measured Keq. (Claims that differences in reductant also affect the data [29] appear to be unfounded, due to control experiments done by Browns group [30].)

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signicant digits). Cob(II)alamin was quantitated by UV Vis spectroscopy (100"5% for all experiments run; lmaxs474 nm, 474s8700 cmy1 My1 [75]). The nucleoside products (from the identical cuvettes used for the kinetic studies, vide infra) were analyzed via HPLC: initial conditions, 95/5 water/acetonitrile, ow 1.0 mL miny1 for 30 min, followed by a gradient to 10/90 water/acetonitrile to clean the column of any contaminants, then a return gradient to 95/5 water/acetonitrile. The disulde product 2,29dithiodiethanol was detected and quantitated by GLC: initial conditions, 75 8C for 3 min, ramp at 5 8C miny1 to 200 8C, then maintained at 200 8C for 8 min. The observed heterolysis (adenine), homolysis (59-deoxyAdo), and disulde products and their yields are given in Table 1 and Scheme 1. 3.4. AdoCbl plus RSH CoC thermolysis kinetic studies First, an appropriate control was done to verify the accuracy of our technique and resultant data: specically, the rate of thermolysis of AdoCbl in H2O without thiol was measured and found to be 6.8(2)=10y5 sy1, in good agreement with our previously published data of 6.5(2)=10y5 sy1 [55]. Kinetic studies of the thermolysis of AdoCbl in the presence of 0.020.1 M b-mercaptoethanol were accomplished by UVVis spectroscopy at 474 nm using the same samples that were used for the product studies (Fig. 4). The data were analyzed by ln[A`/(A`yAt)] versus time plots; a representative plot is shown in Fig. 5, and the results are summarized in Table 1. Temperature-dependence studies were carried out in the same manner, except that the oil-bath temperature was varied from 85 to 110 8C. Spectral and kinetic analyses for these studies are given in Section 4, Figs. AD, along with the derivation of the equations used to obtain the base-on activation parameters. 3.5. AdoCbl plus cysteine thermolysis studies A solution of 4 mL degassed pH 7.0 (0.01 M sodium phosphate buffer) water with 0.020.1 M cysteine was degassed via three freezepumpthaw cycles. In a darkened room using only red light, the solution was added using an airtight syringe to a Schlenk cuvette containing ;0.7 mg AdoCbl under argon to form about a 1=10y4 M solution of AdoCbl. The Schlenk cuvette was then heated in the dark at 100.0"0.2 8C for ;24 h. The reaction was followed by UV Vis spectroscopy at 474 and 522 nm until the reaction was complete (no change in the spectrum at 474 or 522 nm to 3 signicant digits). The nucleoside products, from the identical cuvettes used for the kinetic studies, were analyzed via HPLC: initial conditions, 95/5 water/acetonitrile, ow 1.0 mL miny1 for 30 min, followed by a gradient to 10/90 water/ acetonitrile to clean the column of any contaminants and a return gradient to 95/5 water/acetonitrile. Nucleoside products for 0.1 M cysteine were 95% 59-deoxyAdo and 9% adenine; nucleoside products at -0.1 M cysteine resulted in an incomplete mass balance as discussed in the main text

(0.01 M cysteine yielded 8% adenine, 5% cyclic-Ado, and 61% 59-deoxyAdo, for a 74% mass balance). For [cysteine])0.1 M, a white precipitate formed in the reaction vessel, which was identied as the disulde, cystine, based on decomposition temperature and ESMS results: decomposition at 227228 8C at a heating rate of 1 8C miny1; decomposition point of authentic cystine measured simultaneously gave an identical 227228 8C. Positive ion ESMS calculated mass for [(SCH2CH(NH2)CO2H)2PH]q: 241.3; found parent ion at 241.0. Co(II)Cbl could not be quantitated exactly due to loss of isosbestics after 7 h, although 80(5)% of Co(II)Cbl had formed by ;7 h. The reactions were analyzed by a ln{[AdoCbl]i/[AdoCbl]t} versus time plot until the isosbestics were lost at 7 h. The so-obtained rate constant was independent of thiol concentration: kobss7.7(3)=10y5 sy1 for [cysteine] ranging from 0.002 to 1.0 M. Sample spectra are given in Fig. 7; complete nucleoside product analyses are given in Section 4, Fig. E. 3.6. Attempted measurement of the AdoCbl plus 0.1 M RSH/RSD kH /kD Since the enzymatic work of Stubbe and co-workers [28] requires the assumption that a normal kH/kD-7 isotope effect occurs in the AdoCbl plus protein HS-C408 thiol reaction in RTPR, the solution isotope effect and its temperature dependence, for AdoCbl solution thermolysis in the presence of RSH/RSD, are of interest. In order to accomplish this, the RSH/RSD ratio in aqueous H2O/D2O solution must be quantied. We attempted to do this by dissolving b-mercaptoethanol in predetermined ratios of H2O/D2O (the thiol proton readily exchanges, as seen by the loss of the SH peak in the 1 H NMR in 50/50 H2O/D2O solvent), and then measuring the RSH/RSD ratio directly as a function of temperature by temperature-dependent FT-IR. Unfortunately, and even using attenuated total reectance IR spectroscopy [76], a demonstrated way to reduce water interference [77], the weak RSH (2566 cmy1) and RSD (1863 cmy1) peaks [78] are obliterated by the strong H2O (15001800 cmy1) and D2O (21002800 cmy1) absorbancies [79]. At the normal RSH/ RSD concentrations used in our kinetic studies, 0.1 M thiol, no absorbance for the RSD peak was detectable (no absorbance from 18002000 cmy1 to -0.002 A.U., the noise level), and the absorbance of the RSH peak was very weak (2580 cmy1; absorbance ;0.003 A.U.), ten-fold weaker than the level needed for quantitative studies (;0.03 A.U.). By Beers law, in order to obtain an absorbance of 0.03 A.U. for the RSH peak in a 50/50 RSH/RSD ratio, we would need [thiol]G2.0 M, 20-fold greater thiol concentration than used in the kinetic studies herein. In short, the above data show that it is impossible to obtain the Adox plus RSH/ RSD kH/ kD under the exact conditions (the [thiol] and especially in H2O) that yielded the otherwise near-ideal AdoCbl/RSH/ solvent system studied herein. Development of a system that will allow measurement of the desired kH/kD parameters and as a function of temperature is under further consideration.

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4. Supplementary material Spectral and kinetic analyses of temperature-dependent AdoCbl thermolyses in the presence of b-mercaptoethanol, product analyses for AdoCbl thermolyses in the presence of cysteine, and kinetic derivation for base-on homolysis activation parameters (6 pages) are published in J.M.S.s dissertation available through UMI (J.M. Sirovatka, Colorado State University, Ph.D. Dissertation, 1999). It can also be obtained by contacting the senior author (R.G.F.) directly.

5. Abbreviations AdoCbl Co(II)Cbl Co(III)Cbl BDE DMF 59-DeoxyAdo Cyclic-Ado coenzyme B12; adocobalamin; 59deoxy-59-adenosylcobalamin cob(II)alamin cob(III)alamin bond dissociation energy N,N-dimethylformamide 59-deoxyadenosine 59,8-cyclic deoxyadenosine

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