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 FeIII2-containing protein (∼60%) and the FeIII2-only met-R2 form (∼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 FeIII2 met-R2 component, rate constant 7.4 × 10-3 M-1 s-1 (25 °C) at pH 7.5. The slower second stage is assigned as net reduction of Tyr• and one FeIII of the FeIII2 center, rate constant 1.7 × 10-3 M-1 s-1. Separate experiments with met-R2 protein, and previous evidence from EPR spectroscopy for the formation of an FeIIFeIII intermediate, support such a mechanism. Reduction of the second FeIII is then rapid. The corresponding reduction of the Tyr122Phe R2 variant gives a rate constant of 7.7 × 10-4 M-1 s-1, which is substantially (×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+ (pKa 8.2). Phenylhydrazine (250 mV) is unable to reduce the FeIII2 center, and reacts only with the tyrosyl radical (a 1-equiv process) of the active R2 protein (0.184 M-1 s-1). The reaction is >102 times faster than the 2-equiv N2H4 reduction of Tyr• and FeIII2. The pKa 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.