Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase

Abstract

NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and/or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a(3)/Cu(B)). Reports have variously suggested that during inhibition NO can interact with the binuclear center containing zero (fully oxidized), one (singly reduced), and two (fully reduced) additional electrons. It has also been suggested that two NO molecules can interact with the enzyme simultaneously. We used steady-state and kinetic modeling techniques to reevaluate NO inhibition of CCO. At high flux and low oxygen tensions NO interacts predominantly with the fully reduced (ferrous/cuprous) center in competition with oxygen. However, as the oxygen tension is raised (or the consumption rate is decreased) the reaction with the oxidized enzyme becomes increasingly important. There is no requirement for NO to bind to the singly reduced binuclear center. NO interacts with either ferrous heme iron or oxidized copper, but not both simultaneously. The affinity (K(D)) of NO for the oxygen-binding ferrous heme site is 0.2 nM. The noncompetitive interaction with oxidized copper results in oxidation of NO to nitrite and behaves kinetically as if it had an apparent affinity of 28 nM; at low levels of NO, significant binding to copper can occur without appreciable enzyme inhibition. The combination of competitive (heme) and noncompetitive (copper) modes of binding enables NO to interact with mitochondria across the full in vivo dynamic range of oxygen tension and consumption rates.

Fig. 1.

Hill coefficients for NO inhibition as a function of enzyme turnover. (Left) Hill coefficients (h) obtained from fitting (see Methods) experimental results obtained at 150 μM O₂. Points represent data averaged over intervals of 25 turnover numbers (TNs). Error bars represent SEM; n = 4–12. TN had a significant negative correlation with h (P < 0.05), indicated by the dashed line (although the relationship is unlikely to be strictly linear). (Right) Hill plots of fractional activity as a function of NO at high (Upper) and low (Lower) turnover. The solid line shows the best fit to the experimental data (points) by using nonlinear regression (h = 0.94 and IC₅₀ = 84 nM NO in Upper; h = 1.6 and IC₅₀ = 1,170 nM NO in Lower). For illustrative purposes the data (collected at 1-s resolution) were averaged (±SEM) over 5s.

Fig. 2.

Apparent IC₅₀ for NO as a function of [O₂] at different enzyme TN. (A) Data points represent experimental data: ▴, TN = 13; ▪, TN = 78 (e^– s^–1 aa₃^–1). Error bars represent SEM. Solid lines represent best fit obtained according to the theoretical model proposed by Antunes et al. (43). The optimal rate constants for the fit were obtained by linear regression at the higher TN value. The values of the rate constants obtained were then used to attempt to predict the data at the lower turnover. (Inset) The apparent IC₅₀ for NO as a function of TN at zero oxygen (i.e., y-axis from linear regression with no theoretical constraints). Error bars represent SEM. (B) Here the data are fitted to an expanded model (Fig. 3) in which NO can interact both competitively and noncompetitively with oxygen when the binuclear center is in either the reduced (ferrous a₃) or oxidized (cupric Cu_B) state.

Fig. 3.

A theoretical model that can explain NO inhibition under all conditions of enzyme turnover and oxygen tension. In this model NO can interact both competitively with oxygen and noncompetitively, reacting with the binuclear center in either the reduced (ferrous a₃) or oxidized (cupric Cu_B) state. The equation for IC₅₀ is a rectangular hyperbola, in which the upper value of IC^apparent₅₀ is related to turnover (k_r) (see boxed equation), and K_D′ = k₂/k₁. The fitted values of K_D and K_D′ were 0.2 nM and 28 nM, respectively. The rate of reaction with oxygen (k_o) is 4 * 100 μM^–1, where the factor 4 appears because four electrons are moved to oxygen per reaction step. The internal electron transfer rate k_e is set at 325 s^–1. Values of k_r (initial reduction rate of the binuclear center) are set to accord with the measured turnovers (Fig. 2). Note that, although a number of iron:oxygen intermediates (e.g., ferryl) are also likely to be present during turnover, the interactions of NO with these oxidized species (26, 27) are formally included in the reaction step governed by k₁, and so this added complexity does not affect the conclusions drawn from this model. Additionally, the reaction step governed by k₂ is more complex than indicated because the nitrite dissociation rate depends on the redox state of the binuclear center (29). Thus, K_D′ represents an apparent, not true, affinity constant. (Note that cytochrome c is illustrated bound to CCO to denote the source of electrons, but this does not imply cytochrome c remains bound throughout multiple catalytic cycles.)

Fig. 4.

Apparent IC₅₀ for NO as a function of enzyme turnover at different [O₂]. (A) Points represent experimental data, error bars represent 95% confidence limit, and solid lines are theoretical fits obtained by using the model and associated equation proposed by Antunes et al. (43): IC₅₀ = K_i (1 + k_O2app/k_iv [O₂]), where K_i = 0.2 nM, k_iv is the turnover number in units of e^– s^–1aa₃^–1, and k_O2app was calculated according to Verkhovsky et al. (53). (Inset) the residual obtained after subtracting the theoretical fits from the actual data (•, 150 μM O₂; □, 122 μM O₂; ⋄, 88 μM O₂; ▴, 49 μM O₂). (B) The same data points with solid fit lines corresponding to our model; equation and values are shown in Fig. 3 and its legend (error bars represent SEM). A model in which NO interacts with the binuclear center in both oxidized and reduced states clearly fits the data more closely than does the simple competitive model.

Fig. 5.

Predicted populations of oxidase intermediates and computed turnover as a function of [NO]. The model shown in Fig. 3 was used to generate the predicted populations of oxidized enzyme, NO complexes of oxidized enzyme, and the final NO–ferrous complex. The values shown were obtained for an observed turnover of 15 sec^–1, at 49 μMO₂, using the parameters: k_o = 400 μM; k_r = 15.7 sec^–1; k_e = 325 sec^–1; K_D = 0.2 nM; and K′_D = 28 nM. Population of these species at 49, 88, and 150 μM O₂ can be seen in Fig. 7, which is published as supporting information on the PNAS web site.