Abstract
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.
Highlights
As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b558
Soluble cytochromes from purple phototropic bacteria are almost invariably found to be of the c-type with covalently bound heme [1, 2]
The best characterized soluble cytochrome b is the b562 from Escherichia coli, with a molecular mass of 12 kDa and a redox potential of ϩ200 mV [5,6,7]
Summary
Soluble cytochromes from purple phototropic bacteria are almost invariably found to be of the c-type with covalently bound heme [1, 2]. One of the few previously recognized b-type soluble cytochromes in purple bacteria is a minor component and has a large molecular mass [3]. The best characterized soluble cytochrome b is the b562 from Escherichia coli, with a molecular mass of 12 kDa and a redox potential of ϩ200 mV [5,6,7]. High potential iron protein isozymes were purified from these two species [10], and the sequences were found to be very similar [11].1. They are so similar that the two bacteria may be considered to be strains of the same species. The immediate goal of this study was to identify the cytochrome and to determine its primary and tertiary structures as a contribution to the determination of its functional role
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