Purification and properties of reduced diphosphopyridine nucleotide oxidase from Azotobacter.

Few studies on purified bacterial reduced diphosphopyridine nucleotide (DPNH) or cytochrome oxidases have been reported, although cytochrome reduction by DPNH’ in crude bacterial extracts has repeatedly been described (l-6). Dolin (7, 8) purified a flavoprotein DPNH oxidase and peroxidase from Streptococcus jaecalis, and Lenhoff and Kaplan (9) and Lenhoff et al. (10) described the characteristics of a purified cytochrome peroxidase from Pseudomonas jhorescens. A flavin mononucleotide terminal DPNH oxidase from Escherichia coli was used by Totter and Cormier (11) in luciferase studies; Bruemmer et al. (12) have reported the separation from azotobacter of an electron-transporting particle which possesses a cytochromelinked DPNH oxidase and succinic oxidase; and Tissieres et al. (13) have examined the osidase activities of a small particle fraction of Azotobacter vinelandii. Smith (14) compared the cytochrome oxidase activity of preparations from bacterial and animal sources and found that bacterial preparations oxidized reduced mammalian cytochrome c very slowly. Similarly, when azotobacter cytochrome c and mammalian cytochrome c were tested with their respective oxidases, only the homologous systems oxidized reduced cytochrome c rapidly (3, 4). Cytochrome a2, studied by Tissiirres (15), Moss (16, 17), Chance (I) and Chance et al. (18), forms cyanide and carbon monoxide compounds and is believed to function as the terminal oxidase in such organisms as E. co&, Aerobacter aerogenes, and A. vinelandii, which have no cytochrome a or a3. The present paper describes the separation of a highly active particulate cytochrome-linked DPNH oxidase from A. vinelandii. Cytochromes associated with the DPNH oxidase-containing particle have reduced peaks at 628 to 630 rnp, 590 to 600 rnp, 560 rnp, 552 rnp, 525 to 530 rnp, and 428 rnp, indicating that cytochromes az, al, bl, and cd + cg are present. Examination of the characteristics of the osidase suggested that DPNH may be oxidized by two pathways differing in one or more reactions.