Kinetic studies on the hydrazine and phenylhydrazine reductions of the Escherichia coli R2 subunit of ribonucleotide reductase

Samples of the Escherichia coli R2 protein of ribonucleotide reductase (RNR) normally have two components, the fully active tyrosyl radical (Tyr(.)) and Fe-2(III)-containing protein (similar to 60%) and the Fe-2(III)-only met-R2 form (similar to 40%). Reaction with the 1- or multi- (maximum 4-) equiv reagent hydrazine under anaerobic conditions gives biphasic kinetics. From UV-vis absorbance changes, the first stage of reaction corresponds unexpectedly to reduction of the Fe-2(III) met-R2 component, rate constant 7.4 x 10(-3) M(-1) s(-1) (25 degrees C) at pH 7.5. The slower second stage is assigned as net reduction of Tyr(.) and one Fe-III of the Fe-2(III) center, rate constant 1.7 x 10(-3) M(-1) s(-1). Separate experiments with met-R2 protein, and previous evidence from EPR spectroscopy for the formation of an (FeFeIII)-Fe-II intermediate, support such a mechanism. Reduction of the second Fe-III is then rapid. The corresponding reduction of the Tyr122Phe R2 variant gives a rate constant of 7.7 x 10(-4) M(-1) s(-1), which is substantially (x 10) less than that for met-R2. This is in part explained by the decreased reduction potential of the variant. From pH variations in the range pH 6.6-8.5, N2H4 is the prime reactant with little or no contribution from N2H5+ (pK(a) 8.2). Phenylhydrazine (250 mV) is unable to reduce the Fe-2(III) center, and reacts only with the tyrosyl radical (a l-equiv process) of the active R2 protein (0.184 M(-1) s(-1)). The reaction is > 10(2) times faster than the 2-equiv N2H4 reduction of Tyr(.) and Fe-2(III). The pK(a) for C6H5N2H4+ is 5.27, C6H5N2H3 is the dominant species present under the pH conditions (6.5-8.5) investigated, and no pH dependence is observed. Contrary to a previous report, we conclude that the stability of the diimide (N2H2) does not allow separate studies of the reduction of the R2 protein with this reagent.