Photodissociation and recombination of carbonmonoxy cytochrome oxidase: Dynamics from picoseconds to kiloseconds

Abstract

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 CuB+ at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the CuB(+)-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 microseconds) for the dissociation of CO from the CuB(+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a(3)2+ exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the CuB(+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a(3)2+ (k2 = 1030 s-1). The CO transfer from CuB to Fe alpha 3 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 delta H++ = 10.0 kcal mol-1 and delta S++ = -12.0 cal mol-1 K-1. The value of delta H++ is temperature independent over this range; i.e., delta Cp++ = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a(3)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 microsecond) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by CuB+. We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the CuB(+)-CO complex. We suggest that the picosecond binding of photodissociated CO to CuB triggers the release of a ligand L from CuB. 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the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed.(ABSTRACT TRUNCATED AT 400 WORDS)

The pathway of CO binding to cytochrome c oxidase Can the gateway be closed?

“…The smooth line through the data is a single site binding curve with a Kd of 15.4 mM. This binding constant is relatively weak for hemoprotein-ligand complexes, but is consistent with suggestions of binding constants for the transient complexes of CO [6] and 0, [16] with Cur,.…”

“…[6]). The inset shows a representative plot of the residual differences between the kinetic data obtained here and a single exponential process with a pseudo-first-order rate of 40 s-'.…”

“…The detection system used here is limited to a time resolution of 0.14 ,us, but ns and ps studies are being pursued. In addition, work at high CO pressures to raise the CO solution concentration to the 10 mM range show that a second molecule of CO can, presumably, be bound at the binuclear center [6]. This scheme predicts that such a species would have a much reduced apparent quantum yield relative to the conventional complex with one CO molecule bound at the binuclear center.…”

Observation of the Equilibrium CuB-CO Complex and Functional Implications of the Transient Hemea3 Propionates in Cytochromeba3-CO from Thermus thermophilus

“…2 (traces A-G) the frequency of the CO mode associated with Cu B is invariant between pD 5.5 and 9.7 and unaffected by H/D exchange, consistent with no change in the protonation of groups in close proximity to CO under our experimental conditions. The frequency of (CO) that we observe is similar to those of Limulus hemocyanin ( CO ϭ 2053 cm Ϫ1 ) (27) and nitrite reductase from Alcagenes faecalis ( CO ϭ 2050 cm Ϫ1 ) (30) but significantly different from that found in other Cu B -containing oxidases (14,21,23,26,31,32). In the absence of steric constraints, CO binds to Cu B in a linear fashion (M-C-O), as required for optimal bonding between the d orbitals of Cu and the antibonding * of CO. Back-donation of electron density from the d orbitals to the antibonding * orbitals strengthens the Cu-C bond and weakens the C-O bond.…”

“…where k 1 and k Ϫ1 represent the reversible binding of CO to Cu B , and k 2 is the first-order transfer of CO from Cu B to the heme-Fe (12)(13)(14)(15)(16)(17)(18)(19)(20). The thermal dissociation rate of the heme-CO complex of bovine fully reduced cytochrome c oxidase-CO is very slow (0.023 s Ϫ1 ) and thus, k Ϫ2 can be neglected (13).…”