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

Abstract

Further to a linear free-energy correlation of cross-reaction rate constants k 12 for the reaction of eight organic radicals (OR), e.g. MV + , 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 redox components. The same two techniques of pulse radiolysis and stopped-flow were used. Cross-reaction rate constants (22 °C) at pH 7.0, I=0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E 0 1 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 k 12−0.5 log 10 f versus − E 0 1/0.059 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 is a much stronger oxidant ( E 0∼1.0 V versus NHE) as compared to Fe III 2 ( E 0 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 .