Water-hydroxide exchange reactions at the catalytic site of heme-copper oxidases

Membrane-bound heme-copper oxidases catalyze the reduction of O(2) to water. Part of the free energy associated with this process is used to pump protons across the membrane. The O(2) reduction reaction results in formation of high-pK(a) protonatable groups at the catalytic site. The free energy associated with protonation of these groups is used for proton pumping. One of these protonatable groups is OH(-), coordinated to the heme and Cu(B) at the catalytic site. Here we present results from EPR experiments on the Rhodobacter sphaeroides cytochrome c oxidase, which show that at high pH (9) approximately 50% of oxidized heme a(3) is hydroxide-ligated, while at low pH (6.5), no hydroxide is bound to heme a(3). The kinetics of hydroxide binding to heme a(3) were investigated after dissociation of CO from heme a(3) in the enzyme in which the heme a(3)-Cu(B) center was reduced while the remaining redox sites were oxidized. The dissociation of CO results in a decrease of the midpoint potential of heme a(3), which results in electron transfer (tau approximately equal 3 micros) from heme a(3) to heme a in approximately 100% of the enzyme population. At pH >7.5, the electron transfer is followed by proton release from a H(2)O molecule to the bulk solution (tau approximately equal 2 ms at pH 9). This reaction is also associated with absorbance changes of heme a(3), which on the basis of the results from the EPR experiments are attributed to formation of hydroxide-ligated heme a(3). The OH(-) bound to heme a(3) under equilibrium conditions at high pH is also formed transiently after O(2) reduction at low pH. It is proposed that the free energy associated with electron transfer to the binuclear center and protonation of this OH(-) upon reduction of the recently oxidized enzyme provides the driving force for the pumping of one proton.