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An engineered inter-domain disulfide bridge in flavocytochrome b2 : insights into the role of domain mobility

Flavocytochrome b2 from the yeast Saccharomyces cerevisiae is a Llactate:cytochrome c oxidoreductase. The crystal structure of this homotetrameric enzyme has been solved to 2.4 A (Xia & Mathews, 1990). Each subunit consists of two distinct domains; a small (100 residue) N-terminal cytochrome domain containing a b-type heme, and a larger (411 residue) C-terminal domain containing flavin mononucleotide (FMN). Substrate dehydrogenation occurs at the FMN, which is then re-oxidised by stepwise transfer of electrons via the b2-heme to cytochrome c. The two domains are connected by a hinge sequence, running from residues 89 to 103. It has been proposed that the most likely role of this hinge region is to confer interdomain mobility, allowing movement of the cytochrome domain with respect to the flavin domain (White et al., 1993). This proposal is supported by a number of observations from crystallography (Xia & Mathews, 1990) and NMR spectroscopy (Labeyrie et al., 1988). In light of these observations key questions arise why is there inter-domain mobility in flavocytochrome b2 and is it important for catalysis? Previous experiments involving "hinge-swap" enzyme (White et al., 1993) as well as more subtle mutations involving insertions/deletions to the hinge region (Sharp et a!, 1994, 1996) have illustrated the importance of maintaining the structural integrity of the hinge for efficient inter-domain communication. An alternative approach to examine the importance of domain mobility is to try and restrict it. In this work a disulfide-bridge was engineered in a position, such that the two domains would be fixed in close proximity. Site-directed mutagenesis was used to make the double mutation, N42C:K324C. The crystal structure of the N42C:K324C mutant enzyme was solved to 3.0 A resolution. An inspection of this structure has confirmed the existence of the imposed disulfide-bridge. In addition, the four b2-heme domains of the tetramer are ordered, indicating their limited mobility. Ellman assays were used to chemically confirm the existence of a disulfide-bridge between positions 42 and 324. Steady-state kinetic analyses with L-lactate, using femcyanide [Fe(CN)6} 3 and cytochrome c as electron acceptors were carried out in both the closed (disulfidebridged form) and open (disulfide-bridge cleaved after addition of dithiothreitol)