The methanol : cytochrome c oxidoreductase of methylobacterium extorquens AM1
The methanol : cytochrome c oxidoreductase of methylobacterium extorquens AM1
This thesis reports studies of the electron transfer proteins methanol dehydrogenase (MDH) and cytochrome cL from the facultative methylotroph Methylobacterium extorquens AM1 and investigates the interaction of these proteins. The kinetics of electron transfer between them has been studied using native and chemically modified MDH and the binding of MDH and cytochrome was studied by crosslinking of complexes formed by them. MDH was shown to be tetrameric enzyme with an α_2β2 configuration and not to be dimeric as had previously been concluded. The larger subunit (α-subunit) has a molecular weight of 62 kDa and the newly discovered β-subunit 8.5 kDa. Protein sequencing of the N-terminal of α-subunit and β-subunit showed that they corresponded to the moxF and moxI gene products respectively. Characterisation of cytochrome c-553 from the moxD mutant UV9, showed that it was not cytochrome cL with an uncleaved signal peptide but was a distinct cytochrome c. It has a molecular weight of 23 kDa, a midpoint potential of +194 mV (pH 7.0), is CO reactive, and has an α-absorbance maximum at 553 nm in the reduced state. It is not an electron acceptor from MDH or methylamine dehydrogenase, nor is it a substrate for the oxidase of M.extorquens. Its function in M.extorquens is unknown. A new cytochrome-linked assay for MDH was developd which uses cytochrome c_L to mediate electron transfer to an excess of DCPIP. Ammonium salts were required as activators using this assay which gave higher rates of electron transfer compared with other cytochrome-linked assays. Increasing ionic strength inhibited the reaction indicating that the interaction of MDH and cytochrome is primarily electrostatic. Chemical modification studies in which the charge of lysine residues of MDH were modified showed that the maintenance of the positive charge of lysine residues was essential for activity in the cytochrome-linked assay but was not necessary for activity in the PES-linked assay. Crosslinking using lysine specific heterobifunctional reagents which maintained the positive charge on lysine residues resulted in preferential crosslinking to the β-subunit. Crosslinking using carbodiimides and succinimide esters resulted in crosslinking of cytochrome to both the subunits of MDH. The interaction of MDH and cytochrome cL was shown, by means of a 2-stage EDC/sulpho-NHS enhanced crosslinking reaction, to be primarily by lysine residues of MDH interacting with the carboxyl groups of cytochrome cL and not vice versa.
University of Southampton
1990
Day, Darren John
(1990)
The methanol : cytochrome c oxidoreductase of methylobacterium extorquens AM1.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
This thesis reports studies of the electron transfer proteins methanol dehydrogenase (MDH) and cytochrome cL from the facultative methylotroph Methylobacterium extorquens AM1 and investigates the interaction of these proteins. The kinetics of electron transfer between them has been studied using native and chemically modified MDH and the binding of MDH and cytochrome was studied by crosslinking of complexes formed by them. MDH was shown to be tetrameric enzyme with an α_2β2 configuration and not to be dimeric as had previously been concluded. The larger subunit (α-subunit) has a molecular weight of 62 kDa and the newly discovered β-subunit 8.5 kDa. Protein sequencing of the N-terminal of α-subunit and β-subunit showed that they corresponded to the moxF and moxI gene products respectively. Characterisation of cytochrome c-553 from the moxD mutant UV9, showed that it was not cytochrome cL with an uncleaved signal peptide but was a distinct cytochrome c. It has a molecular weight of 23 kDa, a midpoint potential of +194 mV (pH 7.0), is CO reactive, and has an α-absorbance maximum at 553 nm in the reduced state. It is not an electron acceptor from MDH or methylamine dehydrogenase, nor is it a substrate for the oxidase of M.extorquens. Its function in M.extorquens is unknown. A new cytochrome-linked assay for MDH was developd which uses cytochrome c_L to mediate electron transfer to an excess of DCPIP. Ammonium salts were required as activators using this assay which gave higher rates of electron transfer compared with other cytochrome-linked assays. Increasing ionic strength inhibited the reaction indicating that the interaction of MDH and cytochrome is primarily electrostatic. Chemical modification studies in which the charge of lysine residues of MDH were modified showed that the maintenance of the positive charge of lysine residues was essential for activity in the cytochrome-linked assay but was not necessary for activity in the PES-linked assay. Crosslinking using lysine specific heterobifunctional reagents which maintained the positive charge on lysine residues resulted in preferential crosslinking to the β-subunit. Crosslinking using carbodiimides and succinimide esters resulted in crosslinking of cytochrome to both the subunits of MDH. The interaction of MDH and cytochrome cL was shown, by means of a 2-stage EDC/sulpho-NHS enhanced crosslinking reaction, to be primarily by lysine residues of MDH interacting with the carboxyl groups of cytochrome cL and not vice versa.
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Published date: 1990
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Local EPrints ID: 460522
URI: http://eprints.soton.ac.uk/id/eprint/460522
PURE UUID: b77f3b92-4b47-4047-a8b0-6f11d591f440
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Date deposited: 04 Jul 2022 18:23
Last modified: 04 Jul 2022 18:23
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Author:
Darren John Day
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