The diheme cytochrome c4 from Vibrio cholerae is a natural electron donor to the respiratory cbb3 oxygen reductase

The genome of Vibrio cholerae encodes four respiratory oxygen reductases (1). There are three bd-type oxygen reductases which use ubiquinol as the natural electron donor (2), and one cbb3-type heme-copper oxygen reductase (3), which is presumed to use a cytochrome c as its natural electron donor. The motivation of the current work is to identify the natural electron donor for the cbb3-type heme-copper oxygen reductase. The cbb3-type oxygen reductases are members of the heme-copper superfamily which includes enzymes that perform oxygen reductase chemistry or nitric oxide reduction chemistry (4–7). The vast majority of currently identified oxygen reductases within the heme-copper superfamily are classified as being in one of three families, the A-, B- and C-families (6, 7). The cbb3-type oxygen reductases (7–11) are members of the C-family and represent over 20% of the sequences of heme-copper oxygen reductases from the currently known genome sequences. In the past decade, cbb3-type oxygen reductases have been purified and characterized from several bacterial species, including Rhodobacter sphaeroides (5, 12–17), Paracoccus denitrificans (18), Rhodobacter capsulatus (19–23), Vibrio cholerae (3, 24), Bradyrhizobium japonicum (25–31), Rhodothermus marinus (32), Pseudomonas stutzeri (33–37), Helicobacter pylori (38, 39), and Sulfurihydrogenibium azorense (10). It has been demonstrated by mass spectrometry that the cbb3-type oxygen reductases have the signature histidine-tyrosine cross linked co-factor within their active site, but that the tyrosine comes from a different transmembrane span than for the A-family and B-family heme-copper oxygen reductases (15, 24). The physiological role of the cbb3-type oxygen reductases is generally defined by a high affinity for O2, allowing these enzymes to function at lower oxygen concentrations. Therefore these enzymes are often expressed under microaerophilic growth conditions (9). These enzymes are of particular interest because they are present in a number of pathogenic species and are proposed to be important for virulence by facilitating growth under conditions of low oxygen (40). The cbb3-type enzymes are the only oxygen reductases in Helicobacter pylori (41), Neisseria gonorrhoeae (42) and Neisseria meningitidis (43) and are, therefore, potential drug targets for these significant human pathogens. To understand the physiological roles of the cbb3-type oxygen reductases, it is important to know the natural electron donor(s). In most cases, the natural electron donor is not known. One exception is the cbb3-type oxygen reductase from H. pylori, where the natural electron donor has been shown to be cytochrome c553 (39). When the electron donors are not known, the purified enzymes are usually assayed using the artificial electron donor TMPD (reduced by ascorbate) as the electron donor. In a few instances, reduced horse heart cytochrome c can function as an electron donor (25), though often these assays are also performed in the presence of TMPD. The proteomes of V. cholerae strains have previously been analyzed for the presence of c-type cytochrome binding domains by searching for the canonical sequence CXXCH (44). A total of 48 CXXCH motifs were identified in 31 proteins, of which 14 have N-terminal signal sequences, indicating these are cytochrome c's and are components of the bacterial envelope. Under aerobic growth conditions, six major c-type cytochromes are visualized by SDS-PAGE analysis using a stain for covalently bound heme (44). Two of these six cytochromes c have been identified as the CcoO and CcoP subunits of the cbb3-type oxygen reductase (44). A third heme-containing band was tentatively identified as the YecK subunit of biotin sulfoxide reductase (44). It is likely that one of the heme-staining bands corresponds to the PetC gene, encoding the cytochrome c1 component of the bc1 complex, but this remains to be shown. The best candidates for the remaining two heme-staining bands in aerobically grown V. cholerae are the cycA and cycB gene products, corresponding to cytochrome c4 and cytochrome c5, respectively. Each of these cytochrome c's is predicted to have a cleaved N-terminal signal sequence and to be located within the periplasm. The predicted molecular weights of the mature cytochromes c4 and c5 (19.8 kDa and 11.5 kDa) also make these reasonable candidates for two of the heme-staining bands from aerobically grown V. cholerae (44). The V. cholerae genome also encodes two additional c-type cytochromes of unknown function that are predicted to be periplasmic (44). Their predicted molecular weights (9.1 kDa and 8.5 kDa) do not match the major heme-staining proteins reported for aerobically grown V. cholerae (44). It is likely, therefore, that cytochrome c4 and/or cytochrome c5, might be an electron donor to the cbb3-type oxygen reductase and responsible for shuttling electrons between the bc1 complex and the heme-copper oxygen reductase. For this reason, the genes encoding the di-heme cytochrome c4 and mono-heme cytochrome c5 were each cloned, expressed heterologously in E. coli and the proteins purified. It is shown that cytochrome c5 does not function as an electron donor to the purified cbb3-type oxygen reductase from V. cholerae, whereas cytochrome c4 can support oxygen reductase activity at a rate of at least 300 e−1/s at 25°C.