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

Abstract

NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and͞or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a 3͞CuB). 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 (ferrous͞cuprous) 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.bioenergetics ͉ mitochondria ͉ signaling N O is a physiological signaling molecule produced in vivo by enzymes of the NO synthase family. The NO signaling pathway is classically mediated by activation of soluble guanylate cyclase (1-3). However, in 1994 it was additionally shown that mitochondrial oxygen consumption by cytochrome c oxidase (CCO) is reversibly inhibited by NO in a manner apparently competitive with the oxygen tension (4-6). Inhibition of mitochondrial respiration by NO at CCO has since been implicated in a wide range of physiological processes (7-12), and several of these require a competitive (with respect to oxygen) element to the interaction, e.g., activating hypoxia-inducible factor (13); maintaining flow-metabolism coupling in the brain (14); and allowing cells distant from capillaries to maintain adequate oxygenation (15). Under pathological conditions such as sepsis, the elevated NO concentration, generated by inducible NO synthase, is associated with mitochondrial dysfunction and is implicated in mortality in the critically ill (16).However, the detailed molecular mechanism of CCO inhibition by NO has not yet been unequivocally established. The oxygen-binding center of CCO is bimetallic, and dioxygen reacts at this site only when both the heme a 3 iron and the adjacent copper (Cu B ) are reduced (17). Functional studies initially suggested that NO inhibits by...

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

“…On the other hand, NO reacts with O 2 .− , also generated in the ETC and the ONOO − , which can irreversibly bind or nitrate some of the ETC complexes and attenuate the respiration. It seems that when NO is exogenously added, the complex IV inhibition is significant while the endogenous NO generation leads to ONOO − -induced process oxygen affinity at this site (Table 1), as reported recently by Mason et al that binding at the heme a 3 site depends on the pO 2 (Mason et al 2006). On the other hand, once the pretreated SNAP cells were isolated and re-suspended in the SNAP-free respiratory medium, the Cu B bound NO is known to exert its effect on the CcO activity, in the form of reduced oxygen consumption at a higher pO 2 in comparison to the control (V O 2 max , Table 1).…”

“…by NO and proposed that the binding of endogenous NO to CcO may play a role in diseases such as Parkinson's disease (Cleeter et al 1994). Although the major interaction of NO at complex IV of the ETC is through reversible or irreversible binding at CcO, recent studies have found redox reactions between the bound NO and metal ion centers in CcO, as well as with mitochondrial cytochrome c (Mason et al 2006). Overall, there is a consensus that NO can cause attenuation of cellular respiration and possibly bind at two different metal ion centers of CcO (it is still controversial whether two molecules of NO bind to these sites at the same time or sequentially one NO binds to one of these sites at a given time), namely, the heme a 3 site in competition with O 2 and at the Cu B site where only NO binds, and there is no O 2 binding (non-competitive) (Mason et al 2006).…”

“…Although the major interaction of NO at complex IV of the ETC is through reversible or irreversible binding at CcO, recent studies have found redox reactions between the bound NO and metal ion centers in CcO, as well as with mitochondrial cytochrome c (Mason et al 2006). Overall, there is a consensus that NO can cause attenuation of cellular respiration and possibly bind at two different metal ion centers of CcO (it is still controversial whether two molecules of NO bind to these sites at the same time or sequentially one NO binds to one of these sites at a given time), namely, the heme a 3 site in competition with O 2 and at the Cu B site where only NO binds, and there is no O 2 binding (non-competitive) (Mason et al 2006). To date, it is unclear how the differential NO binding to these two different sites makes a difference in the attenuation of cellular respiration, although it has been well established that NO binding at CcO attenuates respiration.…”

“…4 and Table 1). Based on previous publications, attenuation of V O 2 max can be attributed to NO irreversible binding at the Cu B site and reducing the respiration even at higher existing pO 2 values (Mason et al 2006). Furthermore, it is possible that NO-derived products, such as ONOO − , may induce nitrosation of ETC complexes and cause a reduction in the respiration (Fig.…”

“…ETC inhibition, induced by NO in NOS-stimulated cells, significantly occurs at higher pO 2 values by irreversible inhibition of ETC complexes; yet, the pO 2 -dependent reversible inhibition, which is convincingly demonstrated for exogenously added NO, is not observed. We propose that inhibition of ETC by NO through competitive binding is diminished at low pO 2 ranges due to a lack of excess NO to engage in competitive binding with CcO of complex IV (Mason et al 2006). This mechanism is supported by studying two different systems, in which the NOS function is altered by activation or inhibition.…”