title: 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 k12 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 FeIII2 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, I=0.100 M (NaCl), were determined for the reduction of active-R2 with the eight ORs, reduction potentials E0(1) from -0.446 to +0.194 V. Samples of active-R2 have an FeIII2 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 FeIII2 of met-R2 and then of active-R2. Separate experiments on met-R2 reproduced the first of these rate constants, which on average is approximately 66% larger than the second rate constant. A single Marcus free-energy plot of log k12-0.5 log10f versus -E0(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 (E0 approximately 1.0 V versus NHE) as compared to FeIII2 (E0 close to zero). X-ray structures of the met- and red-R2 states have indicated that electroneutrality of the approximately 10 A 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 FeIII2 component is reduced before the Tyr*.
authors.affiliation: Department of Chemistry, University of Newcastle, Newcastle upon Tyne, UK.
authors.forename: M B
authors.initials: MB
authors.lastname: Twitchett
authors.forename: A M
authors.initials: AM
authors.lastname: Dobbing
authors.forename: A G
authors.initials: AG
authors.lastname: Sykes
mesh_terms: D002021:Buffers; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014443:Tyrosine
chemical_list: D002021:Buffers; D007202:Indicators and Reagents; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit