Interactions of the electron transport proteins of methylotrophic bacteria

Cox, Jonathan Mark (1992) Interactions of the electron transport proteins of methylotrophic bacteria. University of Southampton, Doctoral Thesis.

Abstract

This thesis describes an investigation into the nature of the interactions of the periplasmic electron transport proteins of methylotrophic bacteria. The organisms used in this study were two highly characterised methylotrophs, Methylophilus methylotrophus and Methylobacterium extorquens. Methanol dehydrogenase (MDH) from Methylophilus methylotrophus was shown to contain a small (β) subunit and to exist in an α2β2 subunit conformation. The amino acid sequence of the β-subunit was determined and comparison with the sequence of the same subunit from Methylobacterium extorquens indicated that many of the lysine residues previously proposed to be involved in the reaction with cytochrome c_L were absent. This led to doubts about the role of the β-subunit. The interaction of MDH and the novel c-type cytochrome, cytochrome c_L, was investigated using two cytochrome-linked assay systems, the first using horse-heart cytochrome c as the terminal electron acceptor, the second, a novel assay system developed so as to limit protein/protein interactions to a single site, used the dye PIP as the terminal electron acceptor. Introduction of various salts into the cytochrome-linked assay caused inhibition of electron transport, indicating an interaction that is electrostatic in nature. EDTA was shown to be a potent inhibitor of electron transfer. Modification of the side-chains of various amino acid residues indicated that positively-charged lysine residues on MDH interact with negatively-charged carboxyl groups on cytochrome c_L. Two-stage sulpho-NHS enhanced EDC cross-linking of MDH and cytochrome c_L demonstrated that the cytochrome interacts with the α-subunit of MDH, not with the β-subunit as was originally proposed, and that carboxyl groups on cytochrome c_L cross-link with lysine residues on MDH. Cross-linking was inhibited by the introduction of salts, but not by low concentrations of EDTA, suggesting that EDTA is a potent inhibitor of methanol oxidation by inhibiting electron transfer but not by preventing docking of the two proteins. Two-stage cross-linking also demonstrated that cytochrome c_L interacts with its electron acceptor, cytochrome c_H, in an analogous way to MDH. The reaction is, again, electrostatic in nature, involving carboxyl groups on cytochrome c_L and lysine residues on cytochrome c_H. Competition experiments involving all three proteins indicated that cytochrome c_L possesses a single site that binds either MDH or cytochrome c_H. This is evidence against the `wire-like' theory of electron transfer. Using tuna cytochrome c as a model for cytochrome c_H the residues involved in the EDC/sulpho-NHS cross-link with cytochrome c_L were determined, and were shown to be two of the essential lysine residues involved in the binding of this cytochrome to the reductase and oxidase complexes. A glutamate residue situated close to the haem binding site of cytochrome c_L provides one of the carboxyl groups involved in the cross-link with tuna cytochrome c.

This record has no associated files available for download.

Contributors

Download statistics