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 Ectothiorhodospira 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. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) to be characterized and differs from its eukaryotic counterparts 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 proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue.
Kostanjevecki
V
V
Department of Biochemistry, Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, B-9000 Gent, Belgium.
Leys
D
D
Van Driessche
G
G
Meyer
T E
TE
Cusanovich
M A
MA
Fischer
U
U
Guisez
Y
Y
Van Beeumen
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D002850:Chromatography, Gel; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification; D015786:Cytochromes b5 / Q000737:chemistry*; D020587:Ectothiorhodospira / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019255:NADPH Oxidases; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018629:Spectrometry, Mass, Secondary Ion
D003573:Cytochrome b Group; D011994:Recombinant Proteins; D006418:Heme; D015786:Cytochromes b5; C036888:cytochrome b558; D019255:NADPH Oxidases