ИСТИНА

Subunit I of cytochrome c oxidase (COX) from mitochondria and many bacteria contains a cation binding site located near heme a and facing the P outer aqueous phase. In the bacterial COX it is occupied by tightly bound Ca2+[1,2], whereas the mitochondrial oxidase can bind reversibly Ca2+ or Na+. Until now the role of this center in the enzyme remained obscure. Our previous studies of the effect of sodium concentrations on dissociation constant (Kd) of Ca2+ complex with bovine COX revealed its significant dependence on the redox state of heme a [3]. To clarify the situation we have studied effect of Ca2+ and Na+ on the heme a reduction following 605 nm absorbance band at different redox potentials imposed by ferro/ferricyanide ratio. Media were also supplied by small amount of cytochrome c to facilitate enzyme interaction with iron hexacyanide. At 5mM ferrocyanide and 0.1mM ferricyanide (ca Eh 320 mV) COX depletion of calcium by chelator (0.1mM EGTA) or its substitution by sodium resulted in partial heme a oxidation. Accordingly addition of Ca2+ to calcium depleted enzyme increased heme a reduction. The effect was not observed with Mg 2+ or K+ ions. More detailed oxidative and reductive titrations of bovine COX by ferro/ferricyanide pair have been carried out within range of redox potentials 240-480 mV. In different cases the media contained 0.2mM CaCl2 /0.1mM EGTA, 0.1mM EGTA or 50mM NaCl/0.1mM EGTA. At low redox potential region (240-340mV) titration curves of Ca2+-loaded COX showed much higher levels of heme a reduction confirming previous data that Ca2+ binding to COX stabilizes its reduced state by rising heme a midpoint potential. Such observation might be the first indication of the possible functional role of Ca/Na binding site for cytochrome oxidase. It should be emphathised that no effect on redox titration was observed when Ca2+ was replaced by Mg2+. Also no effect has been found on the oxidative and reductive titration curves upon addition of Ca2+ or chelators to the bacterial COX of wild type from Rhodobacter sphaeroides in which Ca2+ is bound tightly and not removed by complexons [2]. Bacterial COX was kindly provided by Prof.R.Gennis from University of Illinois at Urbana-Champain.