title: 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)
authors.affiliation: Department of Chemistry, University of California, Santa Cruz 95064.
authors.forename: O
authors.initials: O
authors.lastname: Einarsdóttir
authors.forename: R B
authors.initials: RB
authors.lastname: Dyer
authors.forename: D D
authors.initials: DD
authors.lastname: Lemon
authors.forename: P M
authors.initials: PM
authors.lastname: Killough
authors.forename: S M
authors.initials: SM
authors.lastname: Hubig
authors.forename: S J
authors.initials: SJ
authors.lastname: Atherton
authors.forename: J J
authors.initials: JJ
authors.lastname: López-Garriga
authors.forename: G
authors.initials: G
authors.lastname: Palmer
authors.forename: W H
authors.initials: WH
authors.lastname: Woodruff
mesh_terms: D002248:Carbon Monoxide / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D010782:Photolysis; D013057:Spectrum Analysis
chemical_list: D003300:Copper; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV