Biochimica et Biophysica Acta 1995-03-15

Kinetic studies on the reduction of the tyrosyl radical of the R2 subunit of E. coli ribonucleotide reductase.

J C Swarts, M A Aquino, J Y Han, K Y Lam, A G Sykes

Index: Biochim. Biophys. Acta 1247(2) , 215-24, (1995)

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Abstract

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 reductase with hydroxyurea (HU), N-methylhydroxylamine, catechol, and seven hydroxamic acid derivatives are reported. There are no pH-dependences in the range 6.2-8.6 investigated except that introduced with N-methylhydroxylamine 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 with earlier values. Slower reactions are observed with the bulkier acetohydroxamic (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 the protonated form CH3NH2+OH(pKa = 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 observed. Mid-point redox potentials for the reductants and estimates of reduction potentials applying in the case of the protein are considered. The reactions with 1,2-dihydroxybenzene (catechol) and 3,4-dihydroxybenzohydroxamic acid (Didox) also have two stages, when the initial Tyr reduction, rate constants/M-1 s-1 for catechol (3.2) and Didox (0.010), is followed by removal of the Fe(III) to give catechol and catechol like Fe(III)-complexed products. The single stage reactions of the hydroxamic acid derivatives which incorporate charged amino-acid groups 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.