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

Abstract

ABSTRACT The polypeptide and structural gene for a high-molecular-mass c -type cytochrome, cytochrome c 553O , was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c 553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme c 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. capsulatus Bath 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 occ and contains nine c -heme-binding motifs. ORF3 encodes a putative periplasmic c -type cytochrome with a molecular mass of 94,000 Da and contains seven c -heme-binding motifs but shows no sequence homology to occ 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.