High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5′-diphosphate by the E441Q R1 mutant of Escherichia coli ribonucleotide reductase

Abstract

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) cofactor and R1 is the site of the conversion of nucleoside diphosphates to 2-deoxynucleoside diphosphates. A mutant in the R1 subunit of Escherichia coli RNR, E441Q, was generated in an effort to define the function of E441 in the nucleotide-reduction process. Cytidine 5-diphosphate was incubated with E441Q RNR, and the reaction was monitored by using stopped-f low UV-vis spectroscopy and highfrequency (140 GHz) time-domain EPR spectroscopy. These studies revealed loss of the Y⅐ and formation of a disulfide radical anion and present experimental mechanistic insight into the reductive half-reaction catalyzed by RNR. These results support the proposal that the protonated E441 is required for reduction of a 3-ketodeoxynucleotide by a disulfide radical anion. On the minute time scale, a second radical species was also detected by high-frequency EPR. Its g values suggest that this species may be a 4-ketyl radical and is not on the normal reduction pathway. These experiments demonstrate that high-field time-domain EPR spectroscopy is a powerful new tool for deconvolution of a mixture of radical species.Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms (1-3). These enzymes have been divided into four classes based on the metallo-cofactor required to initiate the radical-dependent nucleotide-reduction process. Recent structural studies on class I and class III RNRs (4-6) support the original proposal of Stubbe and coworkers that, despite the differences in the metallo-cofactors in the various classes of RNRs, the function of each cofactor is to generate a thiyl radical that, via a common mechanism, initiates nucleotide reduction by 3Ј-hydrogen atom abstraction (Scheme 1) (7-9). A further commonality between class I and II RNRs is the presence of a conserved glutamate residue adjacent to the two cysteine residues that deliver the reducing equivalents essential for the reduction process (5). This paper provides evidence that the role of this glutamate (in the protonated form) is to facilitate the reduction of the 3Ј-ketodeoxynucleotide 3 (Scheme 1) by a disulfide radical anion intermediate.Much is known about the initial stages of the reduction process (1, 10). There is direct evidence that in the class II, adenosylcobalamin-dependent RNRs, the metallo-cofactor generates a thiyl radical in a kinetically-competent fashion (11,12). Recent studies with a class I RNR containing a diferric tyrosyl radical (Y⅐) cofactor and a class II RNR using cytidine nucleotide analogs provided direct evidence that the thiyl radical generates a 3Ј-nucleotide analog radical (13-16). Once 1 is generated, there is also excellent model precedent that a ketyl radical 2 is generated subsequent to loss of water from the 2Ј position of the nucleotide (17, 18).The mechanism for the reduction of 2 has received less experimental a...

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

“…Recent theoretical studies including E441 in its anionic form predict a free energy barrier of q43.9 kJ/mol and a reaction free energy of q29.7 kJ/mol for this step (Cerqueira et al, 2004b). This proposal is currently only in conflict with the EPR detection of a disulfide radical anion in the reaction of a E441Q R1 mutant (Lawrence et al, 1999), as a glutamine in position 441 would not be able to promote efficient acid/ base-catalyzed dehydration. A way out of this predicament requires an alternative pathway for the E441Q R1 mutant to generate the disulfide radical anion that is independent of the elimination reaction.…”

“…This view is supported by the radical nature of the enzyme (see above), by a battery of results from mechanism-based inhibitor reactions (van der Donk et al, 1995;Covè s et al, 1996;Fontecave, 1998), and by the outcome of studies with isotopically labeled compounds (Stubbe and Ackles, 1980;Stubbe et al, 1983) and with site-directed mutants (Mao et al, 1992a,b,c;Lawrence et al, 1999). Unfortunately, there is still no direct spectroscopic evidence for substrate radicals in the transformation of natural substrates by the wildtype enzyme.…”

“…While the essential residues involved in the catalytic cycle have been identified through activity studies of sitedirected mutants (Mao et al, 1992a,b,c;Lawrence et al, 1999), the proximity of the most relevant residues, Cys 225 , Cys 439 , Cys 462 , Glu 441 , and Asn 437 , to the substrate C29 and C39 centers has been revealed through X-ray crystal structure analysis of the R1 protein with bound substrate (Uhlin and Eklund, 1994;Eriksson et al, 1997;Persson et al, 1997). These pieces of evidence were splendidly merged with results from experiments on small model systems in homogeneous solution, indicating that the elimination of water can indeed be effected via a radical pathway.…”

“…• in protein R2 to generate the putative thiyl radical at C439 in protein R1 (Sjö berg, 1997;Siegbahn et al, 1998;Stubbe et al, 2003); and (iii) catalytic reduction of the bound ribonucleotide to the corresponding deoxyribonucleotide initiated by the thiyl radical C439 Sjö berg, 1997; Persson et al, 1998;Siegbahn, 1998;Lawrence et al, 1999;Stubbe et al, 2003;Zipse, 2003;Kolberg et al, 2004).…”

Resolving the EPR Spectra in the Cytochrome bc 1 Complex from Saccharomyces cerevisiae

“…If the overlapping species have different electronic spin quantum numbers S and m s (e.g., in the case of an organic radical and certain metal centres), they can be distinguished by their different Rabi oscillation (nutation) frequencies [26]. Using the temperature dependence, differences in spin-lattice relaxation times (T 1e ) can also be exploited to distinguish between different species, and it may be possible to suppress the contribution of one species [27] in order to study a second species in greater detail.…”