The Respiratory System of Azotobacter vinelandii

A detailed procedure is described for the isolation of stable respiratory membranes of relatively high phosphorylation efficiency from nitrogen‐fixing cultures of Azotobacter vinelandii (P/O, NADH = 0.90–1.10). Three phosphorylation sites are detectable. These are located at the level of NADH dehydrogenase (Site I; P/2 e 0.45), between the primary dehydrogenases and the cytochromes (Site II; P/2 e 0.53) and on the minor terminal pathway, to the oxygen side of the c ‐type cytochromes (Site III; P/2 e 0.22). Switching of electron flow between the terminal oxidation pathways with low concentrations of KCN and 2‐ n ‐alkyl‐4‐hydroxyquinoline‐ N ‐oxide causes changes in oxygen uptake and phosphate esterification which are compatible with the postulated locations of the three phosphorylation sites and which demonstrate, indirectly, the non‐phosphorylating nature of the b 1 → a 2 major terminal pathway. Phosphorylation efficiences with ascorbate plus purified A. vinelandii cytochromes c 4 and c 5 , horse heart cytochrome c and the artificial electron electron mediators 2,6‐dichlorophenol indophenol and N,N,N′,N′ ‐tetramethyl‐ p ‐phenylene diamine indicate that the minor terminal pathway is further split into a slow, phosphorylating pathway ( c 5 →oxygen) and a faster, non‐phosphorylating pathway ( c 4 →oxygen).