Kinetic properties of ba(3) 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 d ecay of the F intermediate is significantly slower (k' = 100 s(-1) at 5 deg rees C) than in the mitochondrial enzyme, with an activation energy E* of 1 0.1 +/- 0.9 kcal mol(-1). The cyanide-inhibited ba(3) oxidizes cyt c(522) q uickly (k approximate to 5 x 10(6) M-1 s(-1) at 25 degrees C) and selective ly, with an activation energy E* of 10.9 +/- 0.9 kcal mol(-1), but slowly o xidizes ruthenium hexamine, a fast electron donor for the mitochondrial enz yme. Cyt c(552) oxidase activity is enhanced up to 60 degrees C and is maxi mal at extremely low ionic strengths, excluding formation of a high-affinit y 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, t he 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 d issociation from cyt a(3)(2+) of ba(3) (k' = 0.8 s(-1) vs k' = 0.02 s(-1) f or beef heart aa(3) at 20 degrees C). The relevance of these results to ada ptation of respiratory activity to high temperatures and low environmental O-2 tensions is discussed.