FACTORS AFFECTING THE H+ E(-) STOICHIOMETRY IN MITOCHONDRIAL CYTOCHROME-C-OXIDASE - INFLUENCE OF THE RATE OF ELECTRON FLOW AND TRANSMEMBRANE DELTA-PH/

A study is presented of the factors affecting the H+/e(-) stoichiometr y of the proton pump of mitochondrial cytochrome c oxidase, isolated a nd reconstituted in phospholipid vesicles (COV). Under level flow cond itions, i.e., in the absence of a transmembrane Delta mu H+, the H+/e( -) ratio, obtained from spectrophotometric measurements of the initial rates of electron flow and H+ release specifically elicited by cytoch rome c, varied from around 0 to 1, depending on the actual rate of ele ctron flow through the oxidase. At steady state the H+/e(-) ratio for the oxidase was specifically depressed by the transmembrane Delta pH. The study of the H+/e(-) ratio of the pump was complemented by an anal ysis of the redox pattern of cytochrome c, Cu-A, and heme a. From both sets of results and recent structural data from other groups, it is c oncluded that the dependence of the H+/e(-) ratio on the rate of elect ron flow through the oxidase and transmembrane Delta pH is associated with the possible occurrence of two electron transfer pathways in cyto chrome c oxidase, a coupled one (cyt c-->Cu-A-->heme a-->heme a(3)-Cu- B) and a decoupled one (cyt c-->Cu-A-->heme a(3)-Cu-B). The contributi ons of the two pathways, differently affected by kinetics and thermody namic factors, will determine the actual H+/e(-) ratio of the pump. A possible role of heme a in the proton pump and the physiological impli cation of the variable H+/e(-) ratio in the oxidase are discussed.