Role of tyrosine 143 in lactate dehydrogenation by flavocytochrome b2. Primary kinetic isotope effect studies with a phenylalanine mutant

Flavocytochrome b2 catalyzes the oxidation of lactate at the expense of cytochrome c. After flavin (FMN) reduction by the substrate, reducing equivalents are transferred one by one to heme b2, and from there on to cytochrome c. The crystal structure of the enzyme is known at 2.4 angstroms resolution, and specific roles in catalysis have been assigned to active side chains. Tyr143 in particular, located at the interface between the flavodehydrogenase moiety and the heme-binding domain, was thought to take part in substrate binding, as well as to orient the heme-binding domain for efficient electron transfer. A first study of the properties of a Tyr143Phe mutant showed that the major effect of the mutation was to decrease the rate of electron transfer from flavin to heme [Miles, C.S., Rouviere-Fourmy, N., Lederer, F., Mathews, F.S., Reid, G.A., Black, M.T., & Chapman, S.K. (1992) Biochem. J. 285, 187-192]. In the present paper, we focus on the effect of the mutation on catalysis of lactate dehydrogenation. We report the deuterium kinetic isotope effects on flavin reduction as measured with stopped-flow methods and on cytochrome c reduction in the steady-state using L-[2-(2)H]lactate. For the wild-type enzyme, isotope effects on FMN reduction, D[k(F)(red)] and D[k(F)(red)]/K(m), were 7.2 +/- 0.9 and 4.2 +/- 1.3, respectively, and for the Y143F mutant values of 4.4 +/- 0.5 and 3.9 +/- 1.1. were obtained. Calculations, from deuterium isotope effects, of substrate K(d) values, combined with knowledge of k(cat)/k(m) values, lead to the conclusion that Tyr143 does stabilize the Michaelis complex by hydrogen bonding to a substrate carboxylate, as was postulated; but the mutation does not destabilize the transition state more than the Michaelis complex. It is concluded that Tyr143 does not play the role of an acid or an electrophilic catalyst which would stabilize the carbanion-like transition state formed in the initial step of the reaction. Tritium isotope effects were also determined using DL[2-(3)H] lactate and yielded (T)V/K figures of 15.8 +/- 1.7 and 11.3 +/- 0.5 for the wild-type and the Y143F mutant. Analysis of the results supports the idea that the (D)V effects determined for FMN reduction are intrinsic isotope effects values and therefore that the mutation induces a change in the structure of the transition state.