A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine-heart cytochrome-bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome-c reductase activity of either the cholate-solubilized or liposome-reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre-steady-state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome-b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin-promoted oxidant-induced extrareduction of b cytochromes. Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe-S protein. This is interpreted as an interaction of Zn2+ with the 2Fe-2S-cluster region of the Fe-S protein, thus giving rise to inhibition of the reductase activity and of the antimycin-insensitive reduction route of b cytochromes. A Scatchard-plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 x 10(-6) M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.