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

摘要

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-2(III) and Tyr(.) redox components. The same two techniques of pulse radiolysis and stopped-flow were used. Cross-reaction rate constants (22 degrees C) at pH 7.0, 1=0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E-1(0) from -0.446 to +0.194 V. Samples of active-R2 have an Fe-2(III) 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 Fem, of met-R2 and then of active-R2. Separate experiments on met-R2 reproduced the first of these rate constants, which on average is similar to 66% larger than the second rate constant. A single Marcus free-energy plot of log k(12)- 0.5 log(10)f versus - E-1(0)/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)similar to 1.0 V versus NHE) as compared to Fe-2(III) (E-0 close to zero). X-ray structures of the met- and red-R2 states have indicated that electroneutrality of the similar to 10 Angstrom 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-2(III) component is reduced before the Tyr(.). (C) 2000 Elsevier Science Inc. All rights reserved. [References: 40]