Low-temperature flash photolysis studies of cytochrome oxidase and its environment.
The CO-binding kinetics of cytochrome a3, in isolated, detergent-solubilized cytochrome oxidase have been studied by flash photolysis over wide ranges of CO concentration and temperature. The results strongly suggest that CO has an intermediate bound state in its path to the final bound state at the heme iron. In the temperture range 230-273 K in frozen aqueous solutions, the recombination rates depend upon CO concentration; at low CO concentrations the kinetics are biphasic. The rate of the faster process depends upon the detergent concentration, that of the slower process upon the salt concentration. In addition, the faster process depends upon the amount of CO photodissociated. It is concluded that the cytochrome oxidase molecules are aggregated in regions that contain detergent and possibly some lipids. The regions retain considerable fluid character well below the macroscopic freezing point of the solution. The faster phase of the recombination is interpreted as the rebinding of CO molecules that remain in the fluid region after photodissociation. The slower phase would then be due to the migration of some dissociated CO out into surrounding frozen solvent. The non-Arrhenius behavior of both phase probably represents partial melting of the medium; preliminary NMR measurements of mobile protons support this hypothesis. Many of the kinetic features described here are also seen in mitochondria; thus the detergent-solubilized cytochrome oxidase may be a useful model system for the ligand-binding behavior of the enzyme in the mitochondrial membrane.
D002248:Carbon Monoxide / Q000378:metabolism; D003080:Cold Temperature; D003580:Cytochromes / Q000378:metabolism; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D010782:Photolysis; D013816:Thermodynamics
D003580:Cytochromes; D006418:Heme; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV