The respiratory chain of Azotobacter vinelandii. III. The effect of cyanide in the presence of substrates.
(1) Cyanide (100 microM) causes a rapid disappearance of the band (648 nm) of oxidized cytochrome d in particles of Azotobacter vinelandii oxidizing NADH. The rate of disappearance of the band can be related to the rate of inhibition of the oxygen consumption. (2) The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide in the presence of substrates deviate from first-order, indicating that at least two conformations of the enzyme are involved. (3) The rate of binding of cyanide to cytohrrome d increases the larger the rate of turnover of the oxidase. From this it is concluded that cyanide binds preferentially to the enzymically active oxidized conformation of cytochrome d. (4) The instantaneous inhibition of the oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) by cyanide is related neither to the binding of cyanide to cytochrome d as determined spectrophotometrically, nor to the rate of inhibition of the oxidation of NADH. This indicates that different oxidases are involved in the oxidation of NADH and of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), in line with the conclusion of Jones and Redfearn (1967) Biochim. Biophys. Acta 143, 340-353. (5) From experiments in which the change in redox states of cytochrome b1 and c-551 were related to the rate of binding of cyanide to cytochrome d, it is concluded that the cytochromes b1 and d are located in the main pathway to oxygen, whereas cytochrome c-551 functions in the branch via cytochrome o. (6) After prolonged incubation of particles with cyanide in the presence of NADH a residual activity (5%) was found in which a b-type cytochrome is involved. This shows the existence of a third but minor pathway to oxygen.
Kauffman
H F
HF
Laboratory of Biochemistry, B. C. P. Jansen Institute, University of Amsterdam, Plantage Muidergracht 12, Amsterdam, The Netherlands.
Van Gelder
B F
BF
D001205:Ascorbic Acid / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003486:Cyanides / Q000378:metabolism*; D017937:Cytochrome d Group / Q000378:metabolism; D004579:Electron Transport / Q000502:physiology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D001426:Bacterial Proteins; D003486:Cyanides; D009243:NAD; D017937:Cytochrome d Group; D013772:Tetramethylphenylenediamine; D001205:Ascorbic Acid; D010100:Oxygen