Cytochrome C : its role in methylotrophic bacteria

This thesis investigates the role of cytochrome c in methylotrophic bacteria. The majority of the experiments have been performed using two organisms, the facultative methylotroph Pseudomonae AM and the obligate methylotroph Methylophilus, methylotrophue. Neither the concentration of membrane-bound cytochrome c nor the HtJO ratio measured in Pseudomonas AM vary with varying conditions of nutrient-limited growth. The methanol dehydrogenase from Methylophilus methylotrophus has been purified. It is a basic protein with a molecular weight of 120000, consisting of two identical sub units. It requires added methanol for stabilisation. Cyanide has been found to be a linear, competitive inhibitor for the substrate methanol and was able to replace methanol as the stabilising agent. It has been shown that the two major soluble cytochromes c (cytoehromes cH and CL) of Pseudomonas AMI and Methylophilus methylotrophus are completely distinct entities and that the two cytochromes cg are similar, as are the two cytochromes cL. Two distinct soluble cytochromes c have also been found in the facultative methylotrophs hyphomicrobium X and Paracoccus denitrificans. Both the pure, soluble cytochromes c from Methylophilus methylotrophus become reduced at alkaline pH in the absence of added reductant. The first-order kinetics of this process suggest that the reduction is intramolecular and thus one of autoreduction. A mechanism has been proposed to explain this observation. It has been shown that the pure methanol dehydrogenase, in the absence of added methanol, will reduce cytochrome cL, but not cytochrome cH, and it is proposed that methanol dehydrogenase lowers the pH for autoreduction by effecting a conformational change in cytochrome c. It has, however, been possible to demonstrate the methanol-dependent reduction of cytochrome c by methanol dehydrogenase, with concomitant formaldehyde formation, by coupling the reaction to the reduction of exogenous mammalian cytochrome c. By studying a similar reaction in Pseudomonas AMI and Paracoccus denitrificans it has been established that methanol dehydrogenase can only reduce one of the two soluble cytochromes c found in methylotrophs. The methanol dehydrogenase from a particular methylotroph usually only reduces cytochrome c from the same organism. The most important contribution of this thesis is the demonstration that the primary acceptor for electrons derived from the oxidation of methanol by methanol dehydrogenase is one of the two soluble cytochromes c found in methylotrophs.