The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.

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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-A 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., Rouvière-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-[2-2H]lactate. For the wild-type enzyme, isotope effects on FMN reduction, D(kredF) and D(kredF)/Km), 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 Kd values, combined with knowledge of kcat/Km 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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