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

The kinetics of the flash-induced photodissociation and rebinding of c arbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectrosco py at room temperature and by Fourier transform infrared (FTIR) spectr oscopy at low temperature. The binding of photodissociated CO to Cu(B) at room temperature is conclusively established by the TRIR absorpti on at 2061 cm-1 due to the C-O stretching mode of the Cu(B+)-CO comple x. 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 fro m 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 cytochr ome a3(2+) (k2 = 1030 s-1). The CO transfer from Cu(B) to Fe(a3) was f ollowed by CO-FTIR between 158 and 179 K and by UV-vis at room tempera ture. The activation parameters over the temperature range 140-300 K a re 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 temperatur e independent over this range; i.e., DELTAC(p)double dagger = 0 for CO transfer. Rapid events following photodissociation and preceding rebi nding of CO to cytochrome a3(2+) were observed. An increase in the alp ha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initi al femtosecond time-scale events accompanying photodissociation. Subse quently, a decrease is observed in the alpha-band absorbance (t1/2 app roximately 1 mus) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the los s 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 photodis sociated 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 absor bance changes. We have previously suggested that the transfer of L pro duces a transient five-coordinate high-spin cytochrome a3 species wher e the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed. The dissociation o f L from the heme is suggested to be the rate-determining step for tra nsfer of CO from its preequilibrium binding site on Cu(B) to its therm odynamically stable binding site on the heme iron. These findings sugg est an additional feature of the ''ligand shuttle'' role that we previ ously proposed for Cu(B) in the functional dynamics of cytochrome oxid ase. We suggest that the ligand shuttle may occur during the binding o f other small molecules, most notably O2, at the active site and that these ligand-exchange processes may represent a control and coupling m echanism for the electron-transfer and proton-translocation reactions of the enzyme.