Effects of Aeration during Growth of Bacillus stearothermophilus on Proton Pumping Activity Change of Terminal Oxidases

Abstract

The effects of aeration during bacterial growth on the proton translocating activity of the respiratory chain of B. stearothermophilus ATCC 8005, which is stable enough for measurement of the H⁺/O ratio by an oxygen pulse method, were examined. For endogenous and ascorbate-N, N, N', N'-tetramethyl p-phenylene diamine (TMPD) respiration, H⁺/O ratios of around 6 and 2 were obtained using resting cells grown under highly aerated conditions. The values were about 4 and 0 when cells were grown under limited-air conditions. Spectrophotometric and enzyme kinetical analyses revealed that both cytochrome caa₃ and pigment-432 (cytochrome cao) were acting as terminal oxidases, while cytochrome b-558 (corresponding to the “cytochrome o-type oxidase” of the thermophilic bacterium PS3 in the previous paper [Sone, N., Kutoh, E., & Sato, K. (1990) J. Biochem. 107, 597-602]) was mainly serving in the cells grown under limited-air conditions. Measurement of the pH change upon ferrocytochrome c pulse with proteoliposomes reconstituted from the membrane extract of vigorously aerated cells and that of limited-air cells suggested that both cytochrome caa₃ and pigment-432 (cytochrome cao) pump protons, while cytochrome b-558 does not.