High-Molecular-Mass Multi-c-Heme Cytochromes from Methylococcus capsulatus Bath

Methylococcus capsulatus Bath is an obligate methylotroph that utilizes methane as its sole energy and carbon source. As for most other methanotrophs, methane and methanol are the only known growth substrates (6, 30). In methanotrophs, methane is oxidized via a series of two electron steps, with methanol, formaldehyde, and formate as intermediates (6, 30). The reductant for the first, energy-dependent, step is supplied by either NADH or by the respiratory chain, depending on which methane monooxygenase (MMO) is expressed (6, 15, 25, 30, 47, 54, 59, 61). The second, two-electron step, catalyzed by the methanol dehydrogenase, involves the oxidation of methanol to formaldehyde with a c-type cytochrome as an electron acceptor (6, 7, 30, 58). Formaldehyde is either assimilated via the serine or ribulose monophosphate cycle (6, 30) or oxidized to formate by either an NAD+-linked or a dye (i.e., cytochrome b)-linked formaldehyde dehydrogenase or by a tetrahydromethanopterin-methanofuran-mediated pathway (6, 13, 30, 55, 62). Lastly, formate is oxidized to carbon dioxide by an NAD+-linked formate dehydrogenase (34). With the possible exception of an electron donor to the membrane-associated methane monooxygenase (pMMO), c-type cytochromes are known to be involved only in the methanol oxidation step (6, 7, 38). In contrast to the limited role of c-type cytochromes in the oxidation of growth substrates, methanotrophs show complex cytochrome c patterns similar to that observed in the facultative methylotrophs (6, 7, 11, 18, 30, 38, 61–65). For example, seven c-type cytochromes have been purified (5, 63–65), and the structural genes for two additional multiheme cytochromes have been identified (this study) in M. capsulatus Bath. Two of the seven have enzymatic activity; cytochrome c-peroxidase (65) and cytochrome P460 (10, 63), while the remaining five appear to function in electron transfer (5, 61, 63, 64). The complexity of the respiratory systems in methanotrophs provides suggestive evidence that the current biochemical models for methanotrophs underestimate the biochemical capabilities of these organisms. In addition to the known growth substrates, methanotrophs will oxidize or co-oxidize a variety of compounds, depending on the form of MMO expressed (14, 15, 20, 39, 52, 54, 59). Cells expressing the soluble MMO will oxidize straight-chain or branched-chain alkanes or alkenes up to eight carbons long as well as cyclic and aromatic compounds (14, 30, 51, 59). Cells expressing the pMMO will oxidize alkanes and alkenes up to five carbons long but will not oxidize cyclic or aromatic compounds (19, 30, 39, 52). With the exception of methane and, in some cases, methanol, the oxidation of other substrates does not support growth and has been termed co-oxidation. Implicit in the use of the term co-oxidation is that the oxidation provides no metabolic energy. However, some cosubstrates may generate metabolic energy. For example, both MMOs catalyze the energy-dependent oxidation of ammonia to hydroxylamine (16, 47, 63). In M. capsulatus Bath, cytochrome P460 catalyzes the four-electron oxidation of hydroxylamine to nitrite (63). This two-step oxidation of ammonia to nitrite is identical to that observed in nitrifying bacteria, although the enzymes catalyzing the steps have been shown to differ (9, 10, 63). The similar mechanisms of oxidation of ammonia in both groups of bacteria suggest that metabolic energy is obtained during ammonia oxidation in methanotrophs. Whether the oxidation of hydroxylamine also provides reductant for ammonia (or methane) oxidation or whether all four electrons are transferred to the terminal oxidases (21, 62) via cytochrome c′ (64) and cytochrome c555 (5) has not been determined for methanotrophs. In the nitrifying bacterium Nitrosomonas europaea, the four electrons from the oxidation of hydroxylamine are transferred to the tetraheme cytochrome, cytochrome c554, which acts as a redox mediator from hydroxylamine oxidoreductase to both the ammonia monooxygenase and the terminal oxidase (17, 33). In the current study, we present the isolation of an octyl-heme cytochrome, cytochrome c553O. The structural gene for cytochrome c553O was part of a gene cluster containing two other putative high-molecular-mass multiheme cytochromes. The physiological role for these proteins is still unknown. However, one or more of these high-molecular-mass cytochromes appears to be induced by ammonia (10) and may function like cytochrome c554 in N. europaea.