Cytochrome C and methanol oxidation in Pseudomonas AM1
Cytochrome C and methanol oxidation in Pseudomonas AM1
This thesis aims to elucidate the role of cytochrome c in methanol oxidation in the facultative methylotroph Pseudomonas AM1.Two approaches have been used in this work. The first involved measurement of H+/O ratios in both wild-type bacteria and mutant PCT 76 respiring endogenous and exogenous substrates. The results suggest. that there are two proton translocating segements operating during the oxidation of NADH in wild-type bacteria and mutant PCT 76 (lacking cytochrome c) and it is proposed that cytochrome c is not essential for respiration and proton translocation from NADH but only from methanol. The second approach involved the purification of two cytochromes c from methanol-grown Pseudomdnas AM1. Cytochrome cH is a basic protein with a molecular weight of 10960 and an Ea of 294mV. Cytochrome cL is an acidic protein with a molecular weight of 20890 and an Eo of 256mV. Both cytochromes are slowly autoxidisable and able to bind CO.The pH dependence of their midpoint redox potentials suggests that there are two ionising groups affecting the redox potentials of both cytochromes c with pK values of about 3.5 and 5.5 in the oxidised species and 4.5 and 6.5 in the reduced species. The nature of the ionising groups is discussed. Both cytochromes c became reduced at alkaline pH in the absence of any added reductant. The first-order kinetics of this process suggests that the reduction is intramolecular and thus one of autoreduction. A thiol group has been proposed as the intramolecular electron donor during this autoreduction. Both cytochromes c were also reduced at pH 7.0 by pure methanol dehydrogenase in the absence of methanol or ammonium salts and it is proposed that methanol dehydrogenase lowers the pK for autoreduction by effecting a conformational change in the cytochromes. Although the results presented in this thesis do not provide an unequivocal demonstration that cytochrome c interacts directly with methanol dehydrogenase during methanol oxidation in Pseudomonas AM1 they do add to the body of circumstantial evidence that cytochrome c is essential for the oxidation of methanol in methylotrophic bacteria.
University of Southampton
1980
O'Keefe, David Thomas
(1980)
Cytochrome C and methanol oxidation in Pseudomonas AM1.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
This thesis aims to elucidate the role of cytochrome c in methanol oxidation in the facultative methylotroph Pseudomonas AM1.Two approaches have been used in this work. The first involved measurement of H+/O ratios in both wild-type bacteria and mutant PCT 76 respiring endogenous and exogenous substrates. The results suggest. that there are two proton translocating segements operating during the oxidation of NADH in wild-type bacteria and mutant PCT 76 (lacking cytochrome c) and it is proposed that cytochrome c is not essential for respiration and proton translocation from NADH but only from methanol. The second approach involved the purification of two cytochromes c from methanol-grown Pseudomdnas AM1. Cytochrome cH is a basic protein with a molecular weight of 10960 and an Ea of 294mV. Cytochrome cL is an acidic protein with a molecular weight of 20890 and an Eo of 256mV. Both cytochromes are slowly autoxidisable and able to bind CO.The pH dependence of their midpoint redox potentials suggests that there are two ionising groups affecting the redox potentials of both cytochromes c with pK values of about 3.5 and 5.5 in the oxidised species and 4.5 and 6.5 in the reduced species. The nature of the ionising groups is discussed. Both cytochromes c became reduced at alkaline pH in the absence of any added reductant. The first-order kinetics of this process suggests that the reduction is intramolecular and thus one of autoreduction. A thiol group has been proposed as the intramolecular electron donor during this autoreduction. Both cytochromes c were also reduced at pH 7.0 by pure methanol dehydrogenase in the absence of methanol or ammonium salts and it is proposed that methanol dehydrogenase lowers the pK for autoreduction by effecting a conformational change in the cytochromes. Although the results presented in this thesis do not provide an unequivocal demonstration that cytochrome c interacts directly with methanol dehydrogenase during methanol oxidation in Pseudomonas AM1 they do add to the body of circumstantial evidence that cytochrome c is essential for the oxidation of methanol in methylotrophic bacteria.
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Published date: 1980
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Local EPrints ID: 462721
URI: http://eprints.soton.ac.uk/id/eprint/462721
PURE UUID: 9fef9139-e5a7-4ec5-85e1-c960aedaf568
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Date deposited: 04 Jul 2022 19:45
Last modified: 04 Jul 2022 19:45
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Author:
David Thomas O'Keefe
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