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

Abstract

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.

Structure and function of the lanthanide-dependent methanol dehydrogenase XoxF from the methanotroph Methylomicrobium buryatense 5GB1C

“…5a). These regions may interact with the positively charged surface of cytochrome c L for electron transfer [42], although cross-linking studies suggest interactions between lysine residues on MDH and carboxylate groups on cytochrome c L [43, 44]. Alternatively, these disordered residues could require other binding partners for stabilization.…”

Characterisation of a red form of methanol dehydrogenase from the marine methylotroph Methylophaga marina

“…The effects of salts on MDH activities, the modification with 2,4,6-trinitrobenzenesulphohate, and the two-stage sulpho-N-hydroxysuccinimide-enhanced cross-linking with 1-(3-dimethylaminopropyl)-3-ethyicarbodi-imide was as described for Methylobacterium extorquens and Acetobacter methanolicus [3,4].…”

“…In the cytochrome-linked assay system this MDH was remarkably resistant to inhibition by NaCI; no inhibition occurred up to 1.0 M NaCI whereas 10 mM NaCI gives about 50% inhibition with MDHs from other methylotrophs [3,4]. In the cytochrome-linked assay system this MDH was remarkably resistant to inhibition by NaCI; no inhibition occurred up to 1.0 M NaCI whereas 10 mM NaCI gives about 50% inhibition with MDHs from other methylotrophs [3,4].…”

“…A preliminary description of its MDH indicated that, although differing in its substrate specificity and isoelectrie point, it is essentially similar to other MDHs; however, the reaction with cytochrome ct. was not investigated, nor were the subunit structure, or the PQO and calcium contents determined [10]. The system from M. marina is of particular interest because it has been shown recently that the interaction of MDH with cytoehrome c L is strongly inhibited by high ionic strength in Methylophilus methylotrophus, Methylobacteriun~ extorquens and Acetobacter methanolicus; it was concluded from this that the interaction between MDH and cytochrome c L is electrostatic in nature, and it was subsequently demonstrated that this interaction is by way of carboxylate groups on the cytochrome c L and lysyl groups on the MDH a-subunit [3,4]. This raises a number of questions about the system for methanol oxidation in M. marina: does its MDH have the usual a2/~l -, tetrameric structure; does it contain the usual complement of PQQ, does it contain Ca-'+; does it react with a c-type cytochrome corresponding to cytochrome ct.; if so, how do these proteins interact in a periplasm that contains about 400 mM NaCI?…”

“…In Gram-negative methylotrophic bacteria, the first oxidation step during growth on methano~ is catalysed by methanol dehydrogenase (MDH) which is an NAD+-independent quinoprotein that is usually assayed in a dye-linked assay system [1]. The structure of MDH is an at/3 -, tetramer [2][3][4][5], and some MDHs have recently been shown to have a single Ca -'+ per tetramer [5,6]. in the two cases studied there are two molecules of PQQ per tetramer [6,7].…”