Flavocytochrome <i>b</i><sub>2</sub> from the yeast <i>Saccharomyces cerevisiae </i>is a L-lactate:cytochrome <i>c</i> oxidoreductase. The crystal structure of this homotetrameric enzyme has been solved to 2.4 Å. Each subunit consists of two distinct domains; a small (100 residue) N-terminal cytochrome domain containing a <i>b</i>-type heme, and a larger (411 residue) C-terminal domain containing flavin mononucleotide (FMN). 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 inter-domain mobility, allowing movement of the cytochrome domain with respect to the flavin domain. In this work a disulphide-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 2.8 Å resolution. An inspection of this structure has confirmed the existence of the imposed disulfide-bridge. In addition, the four <i>b</i><sub>2</sub>-heme domains of the tetramer are ordered, indicating their limiting mobility. Steady-state kinetic analyses with L-lactate, using ferricyanide [Fe(CN)<sub>6</sub>]<sup>3-</sup> and cytochrome <i>c</i> as electron acceptors were carried out. The formation of the disulfide-bridge causes a 15-fold decrease in <i>k</i><sub>cat</sub> with both electron acceptors. Since [Fe(CN)<sub>6</sub>]<sup>3-</sup> can accept electrons from both the FMN and <i>b</i><sub>2</sub>-heme while cytochrome <i>c</i> can only accept electrons from the <i>b</i><sub>2</sub>-heme this indicates that it is the rate of FMN reduction by L-lactate that is primarily affected by disulphide-bridge formation. Pre-steady-state kinetic analyses with L-lactate are consistent with the steady-state data. The formation of the disulphide-bridge makes it impossible to measure the rate constant for FMN reduction directly while <i>b</i><sub>2</sub>-heme reduction shows a rate constant some 450-fold less than in the open. If flavin to <i>b<sub>2</sub></i>-heme electron transfer is much faster then <i>b</i><sub>2</sub>-heme reduction will be limited by the rate of formation of reduced flavin. Thus, disulfide- bridge formation substantially lowers the rate of FMN reduction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:649726 |
| Date | January 2006 |
| Creators | Drewette, Katy J. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13723 |
Page generated in 0.0014 seconds