Kinetic properties of ba3 oxidase from Thermus thermophilus:: Effect of temperature

The kinetic properties of the ba(3) 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 O-2 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 ba(3) oxidizes cyt c(522) quickly (k approximate to 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 c(552) oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c(522)-ba(3) 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 ba(3) for CO at 20 degrees C (K-eq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa(3) (K-eq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a(3)(2+) of ba(3) (k' = 0.8 s(-1) vs k' = 0.02 s(-1) for beef heart aa(3) at 20 degrees C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O-2 tensions is discussed.