Flavodehydrogenase domain

Fig. 5. Schematic representation of the structure of a flavocytochrome 62 subunit. A, The heme domain B, the flavodehydrogenase domain C, the C-terminal tail D, the hinge region linking the two domains E, the proteolytically sensitive loop F, flavin mononucleotide G, protoheme IX.

Limited amino acid sequence information has shown that long-chain a-hydroxyacid oxidase from rat kidney is also related to these FMN-containing oxidoreductases (55). It is likely that several further members of this family remain to be identified. The flavodehydrogenase domain shows no sequence similarity to the lactate dehydrogenase from bacteria and higher eukaryotes that utilize NAD as a substrate. Yeasts lack such an enzyme and the substrate specificity of flavocytochrome 62 has presumably evolved independently of the NAD-linked dehydrogenases. 

It is quite clear that protein engineering will contribute substantially to future investigations of electron transfer in flavocytochrome 62. To date protein engineering has been used to generate a number of single amino acid substitutions and has allowed the independent expression of the two functionally distinct domains of the enzyme. These two approaches can be readily combined, for example, to express the flavodehydrogenase domain with an active site mutation, thereby sim-plyfing analysis of electron transfer to FMN without interference from the cytochrome domain. 

This leads us to the following conclusion the flavodehydrogenase domain in the native flavocytochrome hi exerts very little influence on the efficiency of the electron transfer between cytochrome b2 and cytochrome c within the complex. This result implies that similar transition states must be achieved in the reactions of cytochrome c with both free and bound cytochrome b2 Consequently, the electron transfer occurs via the same mechanism involving the same pathway through the intra-molecular reaction complex. Thus, in both flavocytochrome b2 - cyt.c and cyt.b2 core - cyt.c complexes, the relative distance and orientation of the heme b2 and heme c planes are optimal to achieve the electron-transfer process. 

The characterisation of the complexation between flavocytochrome b2 and cytochrome c has been the subject of many studies (see for example Short et al., 1998 Daff et al., 1996b and CapeillEre-Blandin, 1995). Work on the anomala flavocytochrome b2, for which there is no crystal structure, led to the conclusions that the cytochrome c binding site involved both the flavodehydrogenase and cytochrome domains (CapeillEre-Blandin and Albani, 1987) and that the complex was stabilised by electrostatic interactions (CapeillEre-Blandin, 1982). It is clear that similar conclusions hold true for the S. cerevisiae enzyme (Daff et al., 1996b) for which the crystal... 

Flavocytochrome hi can be cleaved by controlled proteolysis. Each protomer is folded into two domains having different functions, the L-lactate dehydrogenase, the flavodehydrogenase (FDH)4, a tetramer of molecular mass of 160 kDa, and its electron acceptor, the cytochrome hi core, a monomer of molecular mass of 13 kDa (Gervais et al. 1980), which then acts as a one-electron donor to cytochrome c. ... 

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