INHIBITION OF CYTOCHROME-C-OXIDASE ACTIVITY DURING PROLONGED HYPOXIA

During acute (< 30 min) hypoxia, cellular respiration is independent of the O-2 concentration as long as PO2 remains above a critical value (5-10 Torr). Similarly, state 3 respiration by isolated mitochondria is independent of Po-2 above a critical tension of 2-4 Torr. However, rat hepatocytes demonstrate a reversible suppression of respiration and an increase in NAD(P)H concentration during prolonged (2-24 h), but not acute hypoxia [P. T. Schumacker, N. Chandel, and A. G. N. Augusti. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L395-L402, 1993]. This study tested whether respiration is similarly inhibited in isolated mitochondria exposed to low PO, for prolonged periods and whether cytochrome-e oxidase participates in this response. Coupled rat liver mitochondria were incubated under low oxygen conditions (PO2 < 2 Torr) for 2 h. State 3 respiration after reoxygenation to PO2 = 20 Torr was then compared with the value obtained subsequently at 100 Torr. Using succinate and ADP as substrates, we determined that state 3 respiration at 20 Torr was 61.0 +/- 8.4% of the subsequent value at 100 Torr (P < 0.05). By contrast, control mitochondria reoxygenated to 100 Torr first and 20 Torr subsequently showed no significant difference at the two O-2 tensions (P = NS). When TMPD was used as substrate to supply electrons directly to cytochrome c, respiration at 20 Torr in mitochondria incubated at low PO2 for 2 h was 50.7 +/- 6.0% of the value measured subsequently at 100 Torr. To further test the role of cytochrome-e oxidase in this response, isolated bovine heart enzyme was incubated at PO2 = 20 Torr for 4 h. In the presence of excess substrate, O-2 consumption by the enzyme at PO2 = 20 Torr decreased progressively, reaching a turnover rate of 23.5 +/- 1.3 s(-1) compared with 40.7 +/- 0.9 s(-1) during reoxygenation to 100 Torr (P < 0.001) after 4 h. We conclude that electron transport in rat hepatocyte mitochondria is reversibly inhibited after prolonged exposure to low PO2. This inhibition appears to be mediated by a regulatory effect of molecular oxygen on the catalytic behavior of cytochrome-c oxidase. These findings provide a mechanistic explanation for the reversible decrease in respiration by intact hepatocytes during prolonged exposure to hypoxia.