The oxidation of methylamine by the obligate methylotroph, organism 4025
The oxidation of methylamine by the obligate methylotroph, organism 4025
This thesis investigates the `methylamine oxidase' system of an obligate methylotroph, organism 4025, during growth on methylamine with specific reference to the role of blue copper proteins. Two main approaches have been used: analysis of the electron transport components during growth in a variety of conditions; and in vitro reconstruction experiments using pure proteins. In O2-limited cultures, the concentrations of the periplasmic cytochromes c and the blue copper proteins were dependent on the copper concentration in the growth medium. No blue copper proteins were detected at low copper concentrations although growth on methylamine occurred. The optimal copper concentration for bacteria grown on methylamine (3 mg CuSO4.1-1) was higher than for bacteria grown on methanol (0.5 mg CuSO4.1-1). The highest cell densities of bacteria grown on methylamine were obtained at copper concentrations that also gave the highest cytochrome c and blue copper protein concentrations; the concentration of azurin in methylamine-grown bacteria was higher than that of cytochrome cH. With bacteria grown on methanol, amicyanin was never produced. The highest cell density was obtained at a copper concentration that gave the highest concentration of cytochrome cL; azurin was absent. In bacteria grown on methanol, azurin was only detected at copper concentrations above the optimal value of 0.5 mg CuSO4.1-1. Iron-deficient bacteria grown on methylamine had very low concentrations of cytochrome cL and cH and had no detectable blue copper proteins at the copper concentrations normally used to obtain high cell densities. Pure methylamine dehydrogenase (MADH) reduced amicyanin at a high rate, but not cytochromes cL, cH and azurin. In order to reconstitute (in vitro) a complete `methylamine oxidase' system, the o-type oxidase of organism 4025 was partially purified. It was found to be very similar to the cytochrome co of M. methylotrophus and consisted of cytochromes b(MW 33,000) and c (MW 28,000) and was probably arranged in a 2(b+c) complex. The only electron donors to this cytochrome co were azurin and cytochrome cH. The electron transfer chain from methylamine to oxygen was reconstituted with MADH, amicyanin, azurin (or cytochrome cH) and cytochrome co. Amicyanin was always essential. The rate with azurin (115 electrons. s-1 [mol cyt.co]-1 was greater than with cytochrome cH(31 electrons.s-1[mol cyt.co]-1) In summary, the results in this thesis demonstrate that there are two possible 'methylamine oxidase' systems in organism 4025, depending on the copper concentration. Low copper: MeNH2→MADH→Amicyanin→cyt.c_H→cyt.co→O_2 High copper: MeNH_2→MADH→Amicyanin→azurin→cyt.co→O2 This proposal of an electron transport chain lacking soluble cytochromes c is completely novel. Both those electron transport chains were reconstituted to give sufficient rates to support respiration in bacteria.(DX85712)
University of Southampton
1988
Auton, Kevin Andrew
(1988)
The oxidation of methylamine by the obligate methylotroph, organism 4025.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
This thesis investigates the `methylamine oxidase' system of an obligate methylotroph, organism 4025, during growth on methylamine with specific reference to the role of blue copper proteins. Two main approaches have been used: analysis of the electron transport components during growth in a variety of conditions; and in vitro reconstruction experiments using pure proteins. In O2-limited cultures, the concentrations of the periplasmic cytochromes c and the blue copper proteins were dependent on the copper concentration in the growth medium. No blue copper proteins were detected at low copper concentrations although growth on methylamine occurred. The optimal copper concentration for bacteria grown on methylamine (3 mg CuSO4.1-1) was higher than for bacteria grown on methanol (0.5 mg CuSO4.1-1). The highest cell densities of bacteria grown on methylamine were obtained at copper concentrations that also gave the highest cytochrome c and blue copper protein concentrations; the concentration of azurin in methylamine-grown bacteria was higher than that of cytochrome cH. With bacteria grown on methanol, amicyanin was never produced. The highest cell density was obtained at a copper concentration that gave the highest concentration of cytochrome cL; azurin was absent. In bacteria grown on methanol, azurin was only detected at copper concentrations above the optimal value of 0.5 mg CuSO4.1-1. Iron-deficient bacteria grown on methylamine had very low concentrations of cytochrome cL and cH and had no detectable blue copper proteins at the copper concentrations normally used to obtain high cell densities. Pure methylamine dehydrogenase (MADH) reduced amicyanin at a high rate, but not cytochromes cL, cH and azurin. In order to reconstitute (in vitro) a complete `methylamine oxidase' system, the o-type oxidase of organism 4025 was partially purified. It was found to be very similar to the cytochrome co of M. methylotrophus and consisted of cytochromes b(MW 33,000) and c (MW 28,000) and was probably arranged in a 2(b+c) complex. The only electron donors to this cytochrome co were azurin and cytochrome cH. The electron transfer chain from methylamine to oxygen was reconstituted with MADH, amicyanin, azurin (or cytochrome cH) and cytochrome co. Amicyanin was always essential. The rate with azurin (115 electrons. s-1 [mol cyt.co]-1 was greater than with cytochrome cH(31 electrons.s-1[mol cyt.co]-1) In summary, the results in this thesis demonstrate that there are two possible 'methylamine oxidase' systems in organism 4025, depending on the copper concentration. Low copper: MeNH2→MADH→Amicyanin→cyt.c_H→cyt.co→O_2 High copper: MeNH_2→MADH→Amicyanin→azurin→cyt.co→O2 This proposal of an electron transport chain lacking soluble cytochromes c is completely novel. Both those electron transport chains were reconstituted to give sufficient rates to support respiration in bacteria.(DX85712)
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Published date: 1988
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Local EPrints ID: 460946
URI: http://eprints.soton.ac.uk/id/eprint/460946
PURE UUID: 61d6ecf1-5060-40e2-9d5a-f7904f093b1a
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Date deposited: 04 Jul 2022 18:32
Last modified: 04 Jul 2022 18:32
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Author:
Kevin Andrew Auton
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