Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5)

A soluble cytochrome b(558) from the purple phototropic bacterium Ectothior hodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpect edly, the sequence shows homology to eukaryotic cytochromes b(5). As no pro karyotic homologue had been reported so far, we developed a protocol for th e expression, purification, and crystallization of recombinant cytochrome b (558). The structure was solved by molecular replacement to a resolution of 1.65 Angstrom It shows that cytochrome b(558) is indeed the first bacteria l cytochrome b(5) to be characterized and differs from its eukaryotic count erparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b (5) homologues, including soluble and membrane-bound multifunctional protei ns as well as multidomain enzymes such as sulfite oxidase, fatty-acid desat urase, nitrate reductase, and lactate dehydrogenase. A search of the Mycoba cterium tuberculosis genome showed that a previously unidentified gene enco des a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may pr ovide another example of a bacterial b(5) homologue.