Cytochrome c oxidase maintains mitochondrial respiration during partial inhibition by nitric oxide

Abstract

Nitric oxide (NO), generated endogenously in NO-synthase-transfected cells, increases the reduction of mitochondrial cytochrome c oxidase (CcO) at O2 concentrations ([O2]) above those at which it inhibits cell respiration. Thus, in cells respiring to anoxia, the addition of 2.5 μM L-arginine at 70 μM O2 resulted in reduction of CcO and inhibition of respiration at [O2] of 64.0±0.8 and 24.8±0.8 μM, respectively. This separation of the two effects of NO is related to electron turnover of the enzyme, because the addition of electron donors resulted in inhibition of respiration at progressively higher [O2], and to their eventual convergence. Our results indicate that partial inhibition of CcO by NO leads to an accumulation of reduced cytochrome c and, consequently, to an increase in electron flux through the enzyme population not inhibited by NO. Thus, respiration is maintained without compromising the bioenergetic status of the cell. We suggest that this is a physiological mechanism regulated by the flux of electrons in the mitochondria and by the changing ratio of O2:NO, either during hypoxia or, as a consequence of increases in NO, as a result of cell stress.

Cytochrome c oxidase regulates endogenous nitric oxide availability in respiring cells: A possible explanation for hypoxic vasodilation

“…In the first situation, the [O 2 ] is not critical. We have previously demonstrated that increases in [NO] relative to [O 2 ] that are not yet sufficient to inhibit respiration reduce cytochrome c oxidase and that the recruitment of excess capacity of this enzyme is able to maintain O 2 consumption in the cell (31). Only when the NO:O 2 ratio increases to a point at which this excess capacity is fully used does inhibition of respiration ensue.…”

“…Induced cells were harvested and counted as described in ref. 31, then placed in a water bath at 37°C before measurement in the visible light spectroscopy (VLS) chamber.…”

“…We used N,N,NЈ,NЈ-tetramethyl-p-phenylenediamine (TMPD) and ascorbate to increase the electron flow in cytochrome c oxidase, increasing the turnover of the enzyme while also increasing its reduced state (see ref. 31 and Fig. 5 A Inset).…”

“…29-31. In brief, measured changes in optical attenuation were converted into changes in the redox states of the mitochondrial cytochromes by two multiwavelength linear least-squares fits of their specific absorption coefficients (29). A first-order background was included in the least-squares fitting algorithm to account for nonabsorption changes (31). Calibration and operation of the Clark-type O 2 electrode (Rank Brothers) and the NO electrode (amiNO-700, Innovative Instruments) have also been described (29)(30)(31).…”

“…We have therefore investigated the role of cytochrome c oxidase in the metabolic fate of NO by using our method based on visible light spectroscopy (see refs. [29][30][31]. This allows us to study cell respiration and the redox state of cytochrome c oxidase at the same time as which we monitor O 2 and NO concentrations in cells respiring toward hypoxia in an O 2 -tight chamber.…”