Respiration and Oxidative Phosphorylation in Treponema pallidum

Abstract

Exogenous and endogenously generated reduced pyridine nucleotides caused marked stimulation of O 2 uptake when added to treponemal cell-free extracts, which indicated that terminal electron transport was coupled to the consumption of O 2 . Oxidation of reduced nicotinamide adenine dinucleotide (NADH) was shown to correlate stoichiometrically with O 2 reduction, suggesting that NADH was being oxidized through a mainstream respiratory chain dehydrogenase. Oxygen evolution in treponemal extracts was observed after the completion of O 2 uptake which was stimulated by exogenous NADH and endogenously generated reduced NAD phosphate. Oxygen evolution was inhibited by both cyanide and pyruvate, which was consistent with O 2 release from H 2 O 2 by catalase. The addition of exogenous H 2 O 2 to treponemal extracts caused rapid O 2 evolution characteristic of a catalase reaction. A spectrophotometric assay was used to measure ATP formation in T. pallidum cell-free extracts that were stimulated with NADH. P/O ratios from 0.5 to 1.1 were calculated from the amounts of ATP formed versus NADH oxidized. Phosphorylating activity was dependent on P i concentration and was sensitive to cyanide, N, N ′-dicyclohexylcarbodiimide, and carbonyl cyanide m -chlorophenyl hydrazone. Adenine nucleotide pools of T. pallidum were measured by the firefly luciferin-luciferase assay. Shifts in adenine nucleotide levels upon the addition of NADH to cell-free extracts were impossible to evaluate due to the presence of NAD + nucleosidase. However, when whole cells, previously incubated under an atmosphere of 95% N 2 -5% CO 2 , were sparged with air, ATP and ADP levels increased, while AMP levels decreased. The shift was attributed to both oxidative phosphorylation and to the presence of an adenylate kinase activity. T. pallidum was also found to possess an Mg 2+ - and Ca 2+ -stimulated ATPase activity which was sensitive to N, N ′ -dicyclohexylcarbodiimide. These data indicated a capability for oxidative phosphorylation by T. pallidum.