The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

Abstract

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance 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 'hinge-swap' enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. 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 activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow 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, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

A connecting hinge represses the activity of endothelial nitric oxide synthase

“…The apparent importance of the hinge in controlling NOS catalysis is generally consistent with hinge elements modulating the activity of a number of multidomain redox enzymes (24)(25)(26)(27)(28).…”

Isolation and characterization of the flavin-binding domain of flavocytochrome b2 expressed independently in Escherichia coli

“…The lack of observable electron transfer from the purified flavin-binding domain to the isolated cytochrome domain demonstrates the importance of the interdomain interaction for electron transfer. In the intact enzyme, the cytochrome domain is mobile relative to the flavin domain [4,23,24]. It is likely that the covalent linkage between the two domains serves not only to restrict this mobility, maintaining a high collision frequency, but also to achieve the correct relative orientation of the prosthetic groups for electron transfer.…”

Structural and mechanistic aspects of flavoproteins: electron transfer through the nitric oxide synthase flavoprotein domain

“…This in turn may greatly impact the extent and rate of heme reduction in NOS enzymes. Precedent includes flavocytochrome b 2 , where altering its hinge length caused a 10-fold change in the heme reduction rate [111][112][113][114]. The FMN-FNR subdomain hinge (H1 in Fig.…”