KINETIC-STUDIES ON THE REDUCTION OF THE TYROSYL RADICAL OF THE R2 SUBUNIT OF ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE

Kinetic studies at 25 degrees C, I = 0.100 M (NaCl), on the reduction of the tyrosyl radical of the R2 protein of E. coli ribonucleotide red uctase with hydroxyurea (HU), N-methylhydroxylamine, catechol, and sev en hydroxamic acid derivatives are reported. There are no pH-dependenc es in the range 6.2-8.6 investigated except that introduced with N-met hylhydroxylamine which itself protonates in this range. At pH 7.6 the rate constant (0.46 M(-1) s(-1)) for the HU reaction is in agreement w ith earlier values. Slower reactions are observed with the bulkier ace tohydroxamic (0.020 M(-1) s(-1)) and benzohydroxamic acids (0.040 M(-1 ) s(-1)). In the case of N-methylhydroxylamine the rate constant (0.41 M(-1) s(-1) at pH 7.6) decreases with pH, and it is concluded that th e protonated form CH3NH2+OH (pK(a) = 6.2) has little or no reactivity with Tyr. For this reaction under air-free conditions a second-stage ( 0.027 M(-1) s(-1)) corresponding to reduction of Fe(III)(2) is observe d. Mid-point redox potentials for the reductants and estimates of redu ction potentials applying in the case of the protein are considered. T he reactions with 1,2-dihydroxybenzene (catechol) and 3,4-dihydroxyben zohydroxamic acid (Didox) also have two stages, when the initial Tyr r eduction, rate constants/M(-1) s(-1) for catechol (3.2) and Didox (0.0 10), is followed by removal of the Fe(III) to give catechol and catech ol like Fe(III)-complexed products. The single stage reactions of the hydroxamic acid derivatives which incorporate charged amino-acid group s L-glutamic acid, L-histidine, L-glycine and L-lysine, are slow, and saturation kinetics are observed consistent with association (small K values) prior to redox. The mechanism of reduction of R2-Tyr by all of the reagents studied is discussed.