PHOTODISSOCIATION AND RECOMBINATION OF CARBONMONOXYCYTOCHROME OXIDASE - DYNAMICS FROM PICOSECONDS TO KILOSECONDS

The kinetics of the flash-induced photodissociation and rebinding of carbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectroscopy at room temperature and by Fourier transform infrared (FTIR) spectroscopy at low temperature. The binding of photodissociated CO to Cu(B+) at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the Cu(B+)-CO complex. These measurements yield a first-order rate constant of (4.7 +/- 0.6) X 10(5) s-1 (t1/2 = 1.5 +/- 0.2 mus) for the dissociation of CO from the Cu(B+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a3(2+) exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the Cu(B+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a3(2+) (k2 = 1030 s-1). The CO transfer from Cu(B) to Fe(a3) was followed by CO-FTIR between 158 and 179 K and by UV-vis at room temperature. The activation parameters over the temperature range 140-300 K are DELTAH(double dagger) = 10.0 kcal mol-1 and DELTAS(double dagger) = -12.0 cal mol-1 K-1. The value of DELTAH(double dagger) is temperature independent over this range; i.e., DELTAC(p)double dagger = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a3(2+) were observed. An increase in the alpha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initial femtosecond time-scale events accompanying photodissociation. Subsequently, a decrease is observed in the alpha-band absorbance (t1/2 approximately 1 mus) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by Cu(B+). We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the Cu(B+)-CO complex. We suggest that the picosecond binding of photodissociated CO to Cu(B) triggers the release of a ligand L from Cu(B). We infer that L then binds to cytochrome a3 on the distal side and that this process is directly responsible for the observed alpha-band absorbance changes. We have previously suggested that the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed. The dissociation of L from the heme is suggested to be the rate-determining step for transfer of CO from its preequilibrium binding site on Cu(B) to its thermodynamically stable binding site on the heme iron. These findings suggest an additional feature of the ''ligand shuttle'' role that we previously proposed for Cu(B) in the functional dynamics of cytochrome oxidase. We suggest that the ligand shuttle may occur during the binding of other small molecules, most notably O2, at the active site and that these ligand-exchange processes may represent a control and coupling mechanism for the electron-transfer and proton-translocation reactions of the enzyme.