Methylomirabilis oxyfera” is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gram-negative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of “
. Methylomirabilis oxyfera” putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from “
. Methylomirabilis oxyfera” that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of “
. Methylomirabilis oxyfera” cells and catalyzed methanol oxidation with appreciable specific activity and affinity (
of 10 μmol min
of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from “
. Methylomirabilis oxyfera” is the first representative of type 1 XoxF proteins to be described.