THE IMPORTANCE OF THE INTERDOMAIN HINGE IN INTRAMOLECULAR ELECTRON-TRANSFER IN FLAVOCYTOCHROME-B2

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino a cid sequence of this interdomain hinge is dramatically different in fl avocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the differe nce in kinetic properties between the two enzymes. To probe the import ance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this 'hing e-swap' enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase a s is evident from steady-state experiments with ferricyanide as accept or (only 3-fold less active than wild-type enzyme) and stopped-flow ex periments monitoring flavin reduction (2.5-fold slower than in wild-ty pe enzyme). The major effect of the hinge-swap mutation is to lower dr amatically the enzyme's effectiveness as a cytochrome c reductase; k(c at), for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25-degrees-C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase act ivity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the k(cat) for haem reduction in the hinge-swap enzyme (measured by the stopped-f low method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, includ ing kinetic isotope effects with [2-H-2]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer bet ween the two domains of flavocytochrome b2.