Thiyl radical, ribonucleotide reductase

MECHANISM FIGURE 22-41 Proposed mechanism for ribonucleotide reductase. In the enzyme of . coli and most eukaryotes, the active thiol groups are on the R1 subunit the active-site radical (—X ) is on the R2 subunit and in . coli is probably a thiyl radical of Cys439 (see Fig. 22-40). Steps (T) through are described in the text. 

The most comprehensive set of kinetic studies on AdoCbl homolysis have been performed with AdoCbl-dependent ribonucleotide reductase by Stubbe and coworkers (Licht et al., 1999a Licht et al., 1999b). In this enzyme, AdoCbl is used to generate a thiyl radical on a cysteine residue it is this thiyl radical that abstracts hydrogen from the 3-position of the ribonucleotide to faeilitate reduction at C-2. In the presence of dGTP, an allosteric activator of the enzyme, the enzyme catalyzes the reversible cleavage of AdoCbl and formation of thiyl radical in the absence of substrate. This partial reaction proceeds rapidly enough for it to be mechanistically relevant and provides a system to study AdoCbl homolysis which is both simple and amenable to detailed kinetic analysis. 

Licht, S., Gerfen, G. J., and Stubbe, J., 1996, Thiyl radicals in ribonucleotide reductases. Science 271 477n481. 

Figure 25.11 Ribonucleotide reductase mechanism. (1) An electron is transferred from a cysteine residue on R1 to a tyrosine radical on R2. generating a highly reactive cysteine thiyl radical. (2) This radical abstracts a hydrogen atom from C-3 of the ribose unil. (3) The radica at C-3 releases OH from the C-2 carbon atom. Combined with a proton from a second cysteine residue, the OH is eliminated as water. (4) A hydride ion is transferred from a third cysteine residue with the concomitant formation of a disulfide bond. (5) The C-3 radical recaptures the originally abstracted hydrogen atom. (6) An electron is transferred from R2 to reduce the thiyl radical, which also accepts a proton. The deoxyribonucleotide is free to leave Rl. The disulfide formed in the active site must be reduced to begin another cycle.

Class I E. coli ribonucleotide reductase (RNR) exploits all the PCET variances of Fig. 17.3 in order to catalyze the reduction of nucleoside diphosphates to deoxynu-cleoside diphosphates. This reaction demands radical transport across two subunits and over a remarkable 35 A distance [187,188, 219]. The crystal structures of both R1 and R2 subunits have been solved independently [220-222] and a docking model has been proposed [220]. R2 harbors the diferric tyrosyl radical ( Y122) cofactor that initiates nucleotide reduction by generating a transient thiyl radical ( C439) in the enzyme active site located >35 A away in R1 [223]. Substrate conversion is initiated by a hydrogen atom abstraction (Type A PCET) at the 3 position of the substrate by C439 [192]. 

Several enzymes utilize thiyl radicals for substrate conversion. In the ribonucleotide reductase (RNR) class III, pyruvate formate lyase and benzylsuccinate synthase, cysteine thiyl radicals are generated via hydrogen transfer from cysteine to glycine radicals (Reaction (3.25)) [3, 24-27, 48-52]. 

In all three classes of ribonucleotide reductases, a cysteinyl radical (in the E. coli RNRl sequence at position Cys ) abstracts a hydrogen atom from the C3 position of the carbohydrate moiety of the ribonucleotide substrate [3]. Biomimetic model studies of this enzymatic process were designed, achieving intramolecular hydrogen transfer within a tetrahydrofurane-appended thiyl radical (Scheme 3.4 Reactions (3.27) and (3.28) [75, 76]. 

Ribonucleotide reductase presents an exception to the above mechanism where the working radical is a thiyl derived from an active-site cysteine (C408 in the Lactobacillus Idchmannii enzyme) rather than dAdo [35], Mutation of C408 leads to failure of the mutant enzyme to generate detectable levels of cob(ii)alamin. However, the mutant catalyzes epimerization of AdoCbl that is stereoselectively deuter-ated at the 5 carbon bonded to cobalt [36], This indicates that transient cleavage of the cobalt-carbon bond occurs, but when radical propagation to C408 is precluded, recombination of dAdo and cob(ii)alamin is favored. 

Much less is known about the final step of the pathway, specifically the Bi2-dependent conversion of o( into queuosine. In his review of queuosine biosynthesis, Iwata-Reuyl draws a parallel between the reduction of o( and the reaction performed by ribonucleotide reductases and proposes a mechanism involving a thiyl radical and redox-active disulfide (Figure 36). 

T wo main classes of adenosylcobalamin-activated enzymes function by facilitating the homolytic scission of the Co-C5 to cob(II)alamin and the 5 -deoxyadenosyl radical. The resultant 5 -deoxyadenosyl radical initiates catalysis by abstraction of a hydrogen atom, either from a substrate in the case of the class of enzymes that catalyze radical isomerizations, or by abstraction of a hydrogen atom from Cys408-/3-SH in the active site of ribonucleotide reductase II. The resultant enzymatic thiyl radical initiates the reduction mechanism by abstraction of a hydrogen atom from the ribonucleotide substrate. We shall begin with the isomerization/elimination reactions of adenosylcobalamin. 

Finke has established a chemical precedent for the proposed mechanism for thiyl radical formation in the Bi2-dependent ribonucleotide reductase. Thermolysis of AdoCbl with excess /3-mercaptoethanol under anaerobic conditions yielded 90% Co—C homolysis and 10% heterolysis, as determined by product characterization. The homolysis products were 5 -deoxyadenosine, cob(II)alamin, and the disulfide 2,2 -dithiodiethanol. Kinetic studies established a zero-order dependence on thiol at high [RSH], consistent with rate-limiting Co—C homolysis and formation of a discrete Ado- that subsequently abstracts an H atom from the thiol. Consequently, the... 

With the exception of the cobamide-dependent ribonucleotide reductase, the 5 -deoxyadenosyl radical is indicated to be the direct activator of the substrate. In ribonucleotide reductases, a protein-derived thiyl radical takes this role (see the following text). Accordingly, two vmusual ftinctions would be given to the apoen-zyme in the course of the coenzyme Bi2-catalyzed enzymatic reactions first, it would have to contribute to the activation of the bound coenzyme toward the (reversible) formation of radicals, then it would have to help harness the reactivity of the radical intermediates generated (64). 

The main source of thiyl radicals in cells is expected to be reaction (1), because of the relative abundance of C-H bonds. Some biological radicals are nitrogen-or oxygen-centered, such as the tryptophan (indolyl) radical in DNA photolyase [45], and indolyl and tyrosine (phenoxyl) radicals in ribonucleotide reductase [46-49]. Whether oxygen-centred radicals such as phenoxyl radicals (PhO, e.g. from tyrosine) oxidize GSH by hydrogen transfer ... 

Kolberg M, Bleifuss G, Sjdberg B-M, Graslund A, Lubitz W, Lendzian F, Lassmann G. 2002. Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase. Arch Biochem Biophys 397 57-68. 

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