Methane oxidation to methanol at ambient temperature and pressure is a difficult chemical problem. Methylotrophic bacteria, like Methylosinus trichosporium OB3b, can carry out this reaction using two electrons from an electron donor and molecular oxygen.Cytochrome c-554 from Methylosinus trichosporium OB3b is a redox protein with unknown biological function, that might have an important electron-transfer role. Cytochrome c-554 from Methylosinus trichosporium OB3b exhibits a ferric HALS (highly axial low-spin) signal when subjected to low-temperature electron paramagnetic resonance (EPR) spectroscopy. Spectropotentiometric measurements indicate haem heterogeneity.The EPR spectrum has a typical HALS lineshape with a gmax value of 3.41 at pH 7.0 and pH 8.2. The EPR spectrum changes slightly with pH. The EPR spectrum probably consists of two different low-spin species. The presence of two distinct ferric low-spin species can explain the observed haem heterogeneity.The molecular weight of cytochrome c-554 from Methylosinus trichosporium OB3b has been determined to 12 230 Da by mass spectrometry. The cytochrome is found to contain only one haem group.Optical spectroscopy has determined one axial haem ligand to be methionine.The sequence of the thirty-nine N-terminal amino acids has been determined. This fragment reveals the characteristic haem-c binding motif -CXXCH-, where the cysteines covalently attach the haem group to the protein and the histidine provides one of the axial haem ligands. The amino acid sequence of another twenty-eight residue fragment is also determined. 62% of the amino acid sequence of cytochrome c-554 has been determined.The partial amino acid sequence reveals that this cytochrome is homologous to cytochrome c’s from a diverse range of organisms. An identity of more than 50% is found with two Rhodopseudomonas species. Sequence alignments indicate that the smaller amino acid fragment is located at the C-terminal end of the protein.A putative 3D structure model has been made based on the amino acid sequence and structures of homologous proteins.Cytochrome c-554 was compared with other cytochrome c’s that exhibit HALS EPR signals and have histidine and methionine ligation of the haem iron. No correlation between the geometry of the histidine and methionine ligands and the HALS EPR signal has been established.