Dynamics of the binuclear center of the quinol oxidase from Acidianus ambivalens

We have investigated the kinetic and thermodynamic properties of carbon monoxide binding to the fully reduced quinol oxidase (cytochrome aa(3)) from the hyperthermophilic archaeon Acidianus ambivalens. After flash photolysis of CO from heme a(3), the complex recombines with an apparent rate constant of similar to 3 s(-1), which is much slower than with the bovine cytochrome c oxidase (similar to 80 s(-1)). Investigation of the CO-recombination rate as a function of the CO concentration shows that the rate saturates at high CO concentrations, which indicates that CO must bind transiently to Cu-B before binding to heme as. With the A. ambivalens enzyme the rate reached 50% of its maximum level (which reflects the dissociation constant of the Cu-B(CO) complex) at similar to 13 mu M CO, which is a concentration similar to 10(3) times smaller than for the bovine enzyme (similar to 11 mM). After CO dissociation we observed a rapid absorbance relaxation with a rate constant of similar to 1.4 x 10(4) s(-1), tentatively ascribed to a heme-pocket relaxation associated with release of CO after transient binding to Cu-B The equilibrium constant for CO transfer from Cu-B to heme as was similar to 10(4) times smaller for the A. ambivalens than for the bovine enzyme. The similar to 10(3) times smaller CuB(CO) dissociation constant, in combination with the similar to 10(4) times smaller equilibrium constant for the internal CO transfer, results in an apparent dissociation constant of the heme a(3)(CO) complex which is "only" about 10 times larger for the A. ambivalens (similar to 4 x 10(-3) mM) than for the bovine (0.3 x 10(-3) mM) enzyme. In summary, the results show that while the basic mechanism of CO binding to the binuclear center is similar in the A. ambivalens and bovine (and R. sphaeroides) enzymes, the heme-pocket dynamics of the two enzymes are dramatically different, which is discussed in terms of the different structural details of the A. ambivalens quinol oxidase and adaptation to different living conditions.