Effect of growth conditions on the involvement of cytochrome c in electron transport, proton translocation and ATP synthesis in the facultative methylotroph Pseudomonas AM1

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Abstract
The stoicheiometry of proton translocation, the amounts of cytochromes firmly bound to membranes, and cell yields with respect to succinate and O2 have been measured in the facultative methylotroph Pseudomonas AM1 and in the mutant lacking cytochrome c (mutant PCT76) during carbon-limited growth and carbon-excess growth. →H+/O ratios during endogenous respiration of about 4 were measured in wild-type bacteria grown in carbon-excess conditions, and in the mutant in all growth conditions. During methanol- or succinate-limited growth of wild-type bacteria the →H+/O ratio increased to about 6. Cell yields with respect to succinate and O2 were higher in wild-type than in the mutant lacking cytochrome c by an amount suggesting loss in the mutant of 30% of the ATP-generating capacity of wild-type bacteria. During carbon-limited growth on methanol or succinate some cytochrome c was tightly bound to bacterial membranes, whereas none was tightly bound in bacteria grown in batch-culture or in NH4+-limited conditions. It is proposed that the role of cytochrome c in Pseudomonas AM1 depends on growth conditions and hence on the ‘needs’ of the bacteria. During growth in carbon-excess conditions it is only required for methanol oxidation, mediating between methanol dehydrogenase and cytochrome a/a3. In these conditions oxidation of NADH and succinate by way of cytochrome b and cytochrome a/a3 occurs without the mediation of cytochrome c. This is the only route for oxidation of NADH and succinate in the cytochrome c-deficient mutant in all growth conditions. During carbon-limited growth the cytochrome c becomes bound to the membrane in such a way that it can mediate between cytochromes b and a/a3, hence becoming involved in proton translocation and ATP synthesis during NADH and succinate oxidation. An alternative possibility is that in wild-type bacteria the cytochrome c is always involved in electron transport, but that its involvement in measurable proton translocation only occurs in carbon-limited conditions.