Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature.
The kinetic properties of the ba3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70 degrees C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O2 reaction, the decay of the F intermediate is significantly slower (k' = 100 s-1 at 5 degrees C) than in the mitochondrial enzyme, with an activation energy E of 10.1 +/- 0.9 kcal mol-1. The cyanide-inhibited ba3 oxidizes cyt c522 quickly (k approximately 5 x 10(6) M-1 s-1 at 25 degrees C) and selectively, with an activation energy E of 10.9 +/- 0.9 kcal mol-1, but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c552 oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c522-ba3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba3 for CO at 20 degrees C (Keq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa3 (Keq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a32+ of ba3 (k' = 0.8 s-1 vs k' = 0.02 s-1 for beef heart aa3 at 20 degrees C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O2 tensions is discussed.
Giuffrè
A
A
Department of Biochemical Sciences, CNR Center of Molecular Biology, University of Rome "La Sapienza", Italy.
Forte
E
E
Antonini
G
G
D'Itri
E
E
Brunori
M
M
Soulimane
T
T
Buse
G
G
D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D003573:Cytochrome b Group / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D009994:Osmolar Concentration; D010100:Oxygen / Q000737:chemistry; D011048:Polarography; D012966:Sodium Cyanide / Q000494:pharmacology; D013053:Spectrophotometry; D013057:Spectrum Analysis; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*
D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D002248:Carbon Monoxide; C058557:cytochrome ba3; D003576:Electron Transport Complex IV; D012966:Sodium Cyanide; D010100:Oxygen