The Purification and Characterization of the o-type Cytochrome Oxidase from Methylophilus methylotrophus, and its Reconstitution into a 'Methanol Oxidase' Electron Transport Chain

Abstract

The o-type cytochrome oxidase from methanol-grown Methylophilus methylotrophus has been solubilized and purified (15-fold) to homogeneity. The pure, active oxidase consisted of equal amounts of b-type and c-type cytochromes, corresponding to the two types of protein subunit seen on SDS-PAGE; these had molecular weights of 31 500 and 23800, respectively. The active oxidase probably has two cytochrome c subunits and two cytochrome b subunits, both cytochrome types reacting with CO. The cytochrome c subunit did not correspond to either of the two soluble cytochromes c from M. methylotrophus. The pure oxidase complex oxidized TMPD very rapidly, having a V max of 36 μmol O2 min−1 (mg protein)−1. It was inhibited noncompetitively by azide (K i, 1.13 μM) and KCN (K i, 0.2 μM), the K i values being similar to those measured during respiration by whole bacteria. Of the two soluble cytochromes c from M. methylotrophus, cytochrome c H was oxidized at 50 times the rate of cytochrome c 1, the turnover number with cytochrome c H as substrate being 21 s−1. The cytochrome c component of the oxidase was unable to act as electron acceptor from pure methanol dehydrogenase (soluble, or after solubilization from membranes). A complete ‘methanol oxidase’ electron transport chain was reconstituted from completely pure proteins: methanol dehydrogenase, cytochrome c 1 and c H and the cytochrome c oxidase complex. This ‘methanol oxidase’ showed the same sensitivity to inhibition as observed during methanol oxidation by whole bacteria.