Inhibitors of electron transport in the cytochrome bd complex of Azotobacter vinelandii.

Antimycin A, UHDBT and myxothiazol are representatives of different groups of inhibitors of the mitochondria1 bcl complex where they selectively act on the specific sites for quinone reduction and oxidation. We report here their effects on the purified cytochrome bd terminal oxidase complex from Auinelandii which functions as a quinol oxidase. The quinol binding site is, in the E. coli enzyme, located in subunit I which also contains the hewcoordinate haem b558 111. The oxygen reduction site, comprising haems b595 and d, is located on subunit 11. Electron transport is thought to follow the sequence b558 b595 d 9 121. The cytochrome bd complex was purified from the overproducing A.vinelundii strain MK8 131 by ammonium sulphate precipitation of a lauryl-maltoside solubilised membrane preparation. Respiratory activity was assayed at pH 7.9 in anoxygen-electrode with up to 150 pM ubiquinol-1 plus 1mM DTT or 0.5 mM TMPD plus 2 mM sodium ascorbate as substrates. The purified cytochrome bd complex is inhibited by antimycin A and UHDBT with ubiquinol-1 plus DTT as respiratory susbstrate while the TMPD oxidase activity is unaffected. Myxothiazol has no inhibitory effect with either substrate. Inhibition by antimycinA andUHDBT was found to be non-competitive with Ki=Ki'z (1133) pM and (Z(H5) pM, respectively. TMPD oxidation by the E m f i enzyme has been shown to be uneffected by trypsin digestion of subunit I, a procedure which completely abolishes ubiquinol oxidation Ill. The lack of inhibition of TMPD oxidation by antimycin A and UHDBT in the present experiments confirms the presence of a different electron entry point for the two substrates and suggests that the effect of these inhibitors is on the electron transfer pathway prior to haem b595 and d . In order to further localise the site of inhibition by antimycin A, we have followed the spectral changes of the oxidase at 560 nm during reduction with ubiquinol-1 (Fig. 1). At this wavelength the reduced forms of haem b558 and haem b595 both absorb 141. After addition of substrate to uninhibited enzyme, a period of steady state respiration is observed followed, on anaerobiosis, by a sharp rise in absorbance, to about W90% of the fully reduced spectrum. In antimycin A inhibited samples, the steady state level of haem b reduction is increased, as is,