Kinetic Studies on the Hydrazine and Phenylhydrazine Reductions of the Escherichia coli R2 Subunit of Ribonucleotide Reductase

Abstract

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. T...