The two distinct domains of flavocytochrome b(2) (L-lactate:cytochrome c oxidoreductase, EC 126.96.36.199) are connected by a hinge peptide. Kinetics experiments [White, P., Manson, F.D.C., Brunt, C. E., Chapman, S.K., and Reid, GA. (1993) Biochem. J. 291, 89-94] have illustrated the importance for efficient interdomain electron transfer of maintaining the structural integrity of the hinge. To probe the role of the hinge in a more-subtle manner, we have constructed a mutant enzyme, H Delta 3, which has a three amino acid deletion in the hinge region. Intra- and inter-protein electron transfer within H Delta 3 flavocytochrome b(2) and the H Delta 3:cytochrome c redox complex was investigated by steady-state and stopped-flow kinetics analysis. The H Delta 3 mutant enzyme remains a good L-lactate dehydrogenase, as is evident from steady-state experiments with ferricyanide as electron acceptor (40% less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (15% less active than wild-type enzyme). The global effect of the deletion is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of the H Delta 3 enzyme is manifested at two electron-transfer steps on the catalytic cycle of flavocytochrome b(2). First, the rate of heme reduction has fallen 5-fold in H Delta 3 compared with the wild-type enzyme (from 445 to 91 s(-1)), due to poor interdomain electron transfer from flavin to heme. Second, the rate of cyclochrome c reduction in the steady-state has fallen 5-fold (from 207 to 39 s(-1)), indicating that b(2) heme to cytochrome c electron transfer has also been disrupted. These data; along with the measured kinetic isotope effects, indicate that cytochrome c reduction has become the rate-limiting step in the catalytic cycle for the H Delta 3 enzyme. Further evidence for the importance of the hinge in inter-protein electron transfer is obtained from second-order rate constants for cytochrome c reduction by prereduced flavocytochrome b(2): the rate constant for H Delta 3 is an order of magnitude less than the corresponding value for the wild-type enzyme, with values of 4 x 10(6) and 4.7 X 10(7) M(-1) s(-1), respectively. From our data, we conclude that the hinge plays an important role in facilitating both intra- and inter-protein electron transfer.
- CYTOCHROME C OXIDOREDUCTASE
- L-LACTATE DEHYDROGENASE