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

Abstract

The polypeptide and structural gene for a high-molecular-massc-type cytochrome, cytochromec 553O, was isolated from the methanotrophMethylococcus capsulatus Bath. Cytochromec 553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The hemec concentration was estimated to be 8.2 ± 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c 553O was used to identify a DNA fragment from M. capsulatusBath that contains occ, the gene encoding cytochrome c 553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with occand contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94,000 Da and contains sevenc-heme-binding motifs but shows no sequence homology toocc or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c 553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome.

The presence of multiple c‐type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper

“…The multihaem c-type cytochromes MCA0421 and MCA0423 were abundant at the lower copper concentrations, but showed a fine-tuned regulation of expression between~0 and 1.6 mM copper. Interestingly, and in line with previous reports of purified MCA0423 (Bergmann et al, 1999), our reduced-minusoxidized spectrum showed near identical wavelengths of local maxima of reduced MAC0423. These spectral features may also be representative for other members of the cytochrome c 553o family of proteins.…”

“…Strikingly, the second cysteine in this motif appears to be substituted by histidine in MCA0423, thus disrupting the covalent attachment of haem. In line with this assumption, it has previously been demonstrated that MCA0423 contains eight c-type haem groups (Bergmann et al, 1999), while the other members of the multihaem cytochrome c 553o family are predicted to contain nine. Furthermore, Pfam analyses identified a putative PKD domain N-terminally in MCA0423 and MCA0338 (Fig.…”

“…These spectral features may also be representative for other members of the cytochrome c 553o family of proteins. The genes encoding MCA0421 and MCA0423 are clustered on the M. capsulatus (Bath) genome but appear as two adjacent separate transcriptional units (Bergmann et al, 1999). Importantly, it has not previously been shown that the expression of these proteins depends on the availability of copper.…”

Chloromethane stimulates growth of Methylomicrobium album BG8 on methanol

“…Recently it has been speculated that some of the compounds degraded cometabolically by methane monooxygenase may actually serve as either a carbon or energy source for methanotrophs [4]. To be of any value to the cells, formaldehyde must be made in the oxidation of organic compounds such that the carbon can be converted either into biomass or further oxidized to carbon dioxide to generate more reducing equivalents than that consumed by the methane monooxygenase.…”

The copper responding surfaceome of Methylococccus capsulatus Bath

“…Furthermore, the type and abundance of the particular c‐type cytochrome present were found to vary with the bioavailability of copper ions in the growth medium (Table ) (Karlsen et al ., ). In sMMO‐producing cells, two members of the cytochrome c 553o family were abundant on the M. capsulatus Bath cellular surface [MCA0421 and MCA0423 (denoted occ in Bergmann et al ., )]. Both MCA0421 and MCA0423 are multiheme proteins containing nine and eight c‐type hemes respectively.…”