Mechanism of Oxygen Activation for Tyrosyl Radical Formation in the R2 Subunit of Ribonucleotide Reductase from Mouse
Open Access
- Author:
- Yun, Danny
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 24, 2005
- Committee Members:
- Joseph M Bollinger Jr., Committee Chair/Co-Chair
Squire J Booker, Committee Member
Carsten Krebs, Committee Member
Ming Tien, Committee Member
Michael Thomas Green, Committee Member - Keywords:
- ribonucleotide reductase
oxygen activation
tyrosyl radical - Abstract:
- The R2 subunit of class I ribonucleotide reductase uses a carboxylate-bridged dinuclear iron cluster to activate oxygen for the one-electron oxidation of an internal tyrosine residue. The resulting stable tyrosyl radical is used to generate a cysteine radical in the enzyme's R1 subunit, which in turn generates a substrate 3' radical during nucleotide reduction by the holoenzyme (R1•R2) complex. Extensive mutagenesis, kinetic, and spectroscopic studies of the reaction in the R2 protein from Escherichia coli, which were initiated by the Stubbe group in the early 1990s and carried forward by my predecessors and contemporaries in the Bollinger group, established that (1) acquisition of Fe(II) by the R2 apo protein to form the O2-reactive diiron(II) cluster is relatively slow and rate-limited by a unimolecular step, which is thought to be a conformational change by the protein; (2) addition of O2 to the reactive diiron(II) cluster is fast and produces as the first definitively detectable intermediate a state containing the Fe2(III/IV) complex designated "cluster X" and a cation radical (+•) derived by one-electron oxidation of the near surface tryptophan (W) residue, W48; (3) reduction of the W48+• from solution leaves a state containing only cluster X; and (4) X oxidizes tyrosine 122 to the stable radical (Y122•) in the last and slowest step of the reaction. When I entered graduate school, a key unanswered question regarding the reaction was the nature of precursors to the X-W48+• state, which don't accumulate to levels sufficient for definitive characterization. A µ-(1,2-peroxo)diiron(III) complex was proposed to be one of the precursors on the basis of its accumulation in variants with the Fe1 ligand, D84, substituted by E and the detection of fleeting optical absorption and Mössbauer features similar to those arising from the R2-D84E peroxide complex in the reaction of wild-type Fe(II)-R2 with O2, but definitive evidence for the intermediacy of such a complex in the reaction of a wild-type R2 protein was lacking. Shortly before my enrollment, a study of O2 activation for tyrosyl radical (Y177•) production in R2 from Mus musculus (mouse) had suggested that this reaction might be significantly different from the E. coli R2 reaction in kinetic detail, if not more fundamentally in chemical mechanism [Schmidt, P. P., Rova, U., Katterle, B., Thelander, L, Gräslund, A (1998) J. Biol. Chem. 273, 21463-21472]. The authors had proposed that "mouse X" does not generate Y177• but, rather, is "an intermediate in a side reaction leading to a diferric center without forming the neighboring tyrosyl radical," suggesting that a different intermediate generates the radical in the reaction of this functionally homologous protein. They had further proposed that the slowest step in the mouse R2 reaction is transfer of the electron into the adduct between the diiron(II) cluster and O2, a step that, in the E. coli R2 reaction, is initiated by the rapid (> 400 s-1 at 5 °C) oxidation of W48. This hypothesis assigned to the electron-transfer step in the mouse R2 reaction a rate-constant at least 3300-fold less than that of the cognate step in the E. coli R2 reaction. On the one hand, the hypotheses of Schmidt, et al. implied that conclusions reached by the Bollinger group in previous studies of the E. coli R2 reaction were specific to that R2 protein and reaction, diminishing somewhat their general significance. On the other hand, the proposal that the electron-transfer step is much slower in the mouse R2 reaction implied that preceding intermediates that are "kinetically masked" in the E. coli R2 reaction might accumulate to high levels in the mouse R2 reaction, permitting significant new insight to be extracted from investigation of this system. For these reasons, I decided to re-examine the kinetics and mechanism of O2 activation for Y177• production in mouse R2 as my Ph. D. thesis project. The crucial evidence cited by Schmidt, et al. in support of their hypotheses was the failure of mouse X to accumulate under most reaction conditions and the observation that Y177• is slowed in variants with substitutions to either W103 (the cognate of W48 in E. coli R2) or D266 (the residue that bridges W103 and Fe1 ligand, H173, by hydrogen bonds). In Chapter 2 of this thesis, we show by stopped-flow absorption and freeze-quench EPR and Mössbauer experiments that mouse X does indeed accumulate in the reaction of the Fe(II)-R2 complex with O2, and we present evidence that it is indeed the Y177•-generating intermediate. We further show that generation of Y177• by mouse X is ~ 5-10-fold faster than generation of Y122• by X in the E. coli R2 reaction and that the slowest step in the mouse R2 reaction is acquisition of Fe(II) by the apo protein to form the O2-reactive diiron(II) cluster. In Chapter 3, we present evidence that a peroxodiiron(III) complex that is indistinguishable spectroscopically from the complex characterized in the reactions of D84E variants of E. coli R2 accumulates in the mouse R2 reaction and is probably a precursor to X. These experiments provide the most definitive evidence yet for the intermediacy of this complex in the reaction of a wild-type R2 protein. In addition, the experiments resolve the rate constant for the electron transfer step during formation of X as either ~ 80 or ~ 110 s-1, further disproving the notion of rate-limiting electron transfer. In Chapter 4, we show that the slowest step in Y177• production in the reaction of the Fe(II) complex of R2-W103Y, one of the variants studied by Schmidt, et al., with O2 is addition of O2 to the diiron(II) cluster rather than the electron-transfer step. The results of this chapter debunk the final remaining evidence for the notion of slow electron transfer in the reaction of mouse R2. They show that the primary effect of the W103Y substitution is the retardation of a step in which the residue in question has no obvious chemical role, presumably by a secondary effect on the structure or dynamics of the protein. They thus provide a stark admonition that reactions of variant proteins must be thoroughly characterized for proper interpretation of the effect of the substitution. Together, the results of this thesis imply that O2 activation for tyrosyl radical formation in mouse R2 proceeds by a mechanism much more similar to that of the E. coli R2 reaction than was indicated by the study of Schmidt, et al. They thus consolidate our understanding of O2 activation by R2 specifically and by the diiron-carboxylate proteins in general.