The interaction of methanol dehydrogenase and its cytochrome electron acceptor

A fluorescence method is described for direct measurement of the interaction between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. This has permitted a distinction to be made between factors affecting electron transfer and those affecting the initial binding or docking process. It was confirmed that the initial interaction is electrostatic, but previous conclusions with respect to the mechanism of EDTA inhibition have been modified. It is proposed that the initial ‘docking’ of MDH and cytochrome cL is by way of ionic interactions between lysyl residues on its surface and carboxylate groups on the surface of cytochrome cL. This interaction is not inhibited by EDTA, which we suggest acts by binding to nearby lysyl residues, thus preventing movement of the ‘docked’ cytochrome to its optimal position for electron transfer, which probably involves interaction with the hydrophobic funnel in the surface of MDH.

The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

Quaternary structure of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa and its reoxidation with a novel cytochrome c from this organism

Quinoprotein (2,7,9-tricarboxy-1H-pyrrolo-[2,3-f]quinoline-4,5-dione quinone form (PQQ)-containing) ethanol dehydrogenase (EDH) from Pseudomonas aeruginosa ATCC 17933 was purified to homogeneity. EDH has an alpha 2 beta 2 configuration and subunits comparable in size to those of methanol dehydrogenase (MDH). Compared with other PQQ-containing dehydrogenases, Ca2+ is rather loosely bound and it seems necessary for PQQ binding and stability of EDH. Two soluble cytochromes c were detected in extracts from ethanol-grown cells and both were purified. One of these has an alpha-band at 551 nm for its reduced form, the oxidized form being an excellent electron acceptor for the semiquinone form of EDH. Since this cytochrome is quite different from the already known cytochrome c551 (operating in nitrate respiration) of this organism, it is indicated here as cytochrome cEDH. Comparison of the N-terminal amino acid sequence of cytochrome cEDH with the complete sequence of cytochrome cL (the electron acceptor of MDH), cytochrome cH (the electron acceptor of cytochrome cL) and cytochrome c551 revealed some similarity only to internal stretches of amino acids of the last two. The other soluble cytochrome appeared to be the already-known cytochrome c556. Since it was not an electron acceptor for cytochrome cEDH (neither for EDH), cytochrome cH is lacking in the quinoprotein-EDH-ethanol oxidation system of P. aeruginosa. It seems, therefore, that the respiratory chains for MDH and EDH are different.

The interaction of methanol dehydrogenase and cytochrome cL in the acidophilic methylotroph Acetobacter methanolicus

The quinoprotein methanol dehydrogenase (MDH) of Acetobacter methanolicus has an alpha 2 beta 2 structure. By contrast with other MDHs, the beta-subunit (approx. 8.5 kDa) does not contain the five lysine residues previously proposed to be involved in ionic interactions with the electron acceptor cytochrome cL. That electrostatic interactions are involved was confirmed by the demonstration that methanol:cytochrome cL oxidoreductase activity was inhibited by high ionic strength (I), the strength of interaction being inversely related to the square root of I. Specific modifiers of arginine residues on MDH inhibited this reaction but not the dye-linked MDH activity. Modification of lysine residues on MDH that altered its charge had no effect on the dye-linked activity but inhibited reaction with cytochrome cL. When the charge was retained on modification of lysine residues, little effect on either activity was observed. Cross-linking experiments confirmed that lysine residues on the alpha-subunit, but not the beta-subunit, are involved in the ‘docking’ process between the proteins.

The nucleotide sequence and deduced amino acid sequence of the cytochrome cL gene of Methylobacterium extorquens AM1, a novel class of c-type cytochrome

The nucleotide sequence and deduced amino acid sequence of the cytochrome cL of Methylobacterium extorquens (Pseudomonas AM1; Methylobacterium AM1) shows that this cytochrome c is completely different, except for its haem-binding site, from all other cytochromes.