Dobbing AM

Sykes AG

Twitchett MB

Further to a linear free-energy correlation of cross-reaction rate constants k12 for the reaction of eight organic radicals (OR), e.g. MV(\ub7+) from methyl viologen, with cytochrome c(III), we consider here similar studies for the reduction of the R2 protein of Escherichia coli ribonucleotide reductase, which has Fe(III)2 and Tyr(\ub7) redox components. The same two techniques of pulse radiolysis and stopped-flow were used. Cross-reaction rate constants (22 \ub0C) at pH 7.0, I = 0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E01 from -0.446 to +0.194 V. Samples of active-R2 have an Fe(III)2 met-R2 component, which in the present studies was close to 40%. Concurrent reactions have to be taken into account for the five most reactive ORs, corresponding to reduction of the Fe(III)2 of met-R2 and then of active-R2. Separate experiments on met-R2 reproduced the first of these rate constants, which on average is ~66% larger than the second rate constant. A single Marcus free-energy plot of log k12-0.5 log10 f versus -E01/0.059 describes all the data and the slope of 0.54 is in satisfactory agreement with the theoretical value of 0.50. Such behaviour is unexpected since the Tyr(\ub7) is a much stronger oxidant (E0 ~ 1.0 V versus NHE) as compared to Fe(III)2 (E0 close to zero). X-ray structures of the met- and red-R2 states have indicated that electroneutrality of the ~ 10 Å buried active site is maintained. Proton transfer is therefore proposed as a rapid sequel to electron transfer. Other reactions considered are the much slower conventional time-range reductions of active-R2 with hydrazine and dithionite. For these reactions one and/or two-equivalent changes are possible. With both reductants, met-R2 reacts about four-fold faster than active-R2, and as with the ORs the less strongly oxidising Fe(III)2 component is reduced before the Tyr(\ub7). (C) 2000 Elsevier Science Inc

New mechanistic insights into the reactivity of the R2 protein of E. coli ribonucleotide reductase (RNR)

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