Nitric Oxide Reacts with the Single-electron Reduced Active Site of Cytochrome c Oxidase

Giuffrè, Alessandro; Barone, Maria Cecilia; Brunori, Maurizio; D’Itri, Emilio; Ludwig, Bernd; Malatesta, Francesco; Müller, Hans‐Werner; Sarti, Paolo

doi:10.1074/jbc.m201514200

Cited by 32 publications

(24 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The rapid inhibition of the oxidase by NO also suggested that it may bind an intermediate in the catalytic cycle unavailable to oxygen, making it a more effective inhibitor than predicted thermodynamically by allowing it to ''outcompete'' oxygen kinetically (41). Nevertheless, we suggested that the steady-state inhibition constants observed in vivo are broadly consistent with simple equilibrium NO binding to the same enzyme intermediate as oxygen binds, namely, the fully reduced binuclear center (7,42).…”

supporting

confidence: 52%

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

Mason

Nicholls

Wilson

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

216

158

View full text Add to dashboard Cite

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...

show abstract

supporting

confidence: 52%

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

Mason

Nicholls

Wilson

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

216

158

View full text Add to dashboard Cite

show abstract

“…4 we present an alternate reaction scheme for the high electron flux process that we think satisfactorily accounts for the observations from all laboratories. The present work and that of others clearly support a marked preference of NO for the heme site in variously prepared derivatives, and in transient kinetics experiments the inhibited state is thought to be associated with the formation of nitrosylferroheme a 3 (5,16). Therefore, in the chain of events where NO enters the active site pocket before O 2 , the available evidence strongly suggests that it will bind to heme a 3 (I).…”

Section: Discussionmentioning

confidence: 64%

Reversal of Cyanide Inhibition of Cytochrome c Oxidase by the Auxiliary Substrate Nitric Oxide

Pearce

Bominaar

Hill

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Nitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a 3 by NO with reduction of the heme. The resulting nitrosylferroheme a 3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only ϳ50% in brain mitochondria.The reactions of cytochrome c oxidase with nitric oxide (NO) have recently been the subject of renewed scrutiny (1-6) because of their emerging physiological significance (7-12). Furthermore, the discovery and functional characterization of a mitochondrial NO synthase (9, 13, 14) raises a series of questions regarding the possible regulation of the electron transport chain by NO. Several catalytic cycles have been proposed (4,6,(15)(16)(17) to explain the consumption of NO by the oxidase while turning over in the presence of cytochrome c and oxygen. Some of these cycles implicitly treat the NO as an auxiliary substrate rather than simply a competitive inhibitor, to account for the observed production of nitrite in addition to the slowing of electron transfer rate. In some cases an oxyferryl intermediate of heme a 3 is proposed and interaction of incoming NO with Cu B assumed (15, 16), whereas other schemes invoke the formation of a nitrosylheme a 3 intermediate (4,6,17). Consequently, clarification of which (if either) of the two centers of the binuclear pair, heme a 3 or Cu B , preferentially reacts with NO to form detectable derivatives should be of some value in assessing the relative likelihood of one proposed scheme over another as conditions are varied.When metalloenzymes have multiple binding sites for small molecular substrates and inhibitors, especially within the same active site domain, studies with mixed-ligand adducts can often reveal useful details of the coordination chemistry. Here we report the results of an investigation into the preferred site of NO reaction with bovine heart cytochrome c oxidase in the presence of the potent competitive inhibitor, cyanide, both under conditions of turnover and where the enzyme has been allowed to equilibrate in the presence of NO, cyanide, and reducing equivalents. The findings we report appear to be ...

show abstract

“…4 [16], the relative concentration of reduced intermediates (R and E) tends to rise at higher reduced cytochrome c concentrations, whereas intermediates P and F prevail at lower concentrations. Interestingly, the half-reduced intermediate E seems to react with NO [37]. Thus at higher reductant concentration, reduced cytochrome a 3 is the primary target with formation of a stable nitrosylated adduct (a 3 2+ -NO), whereas at lower reductant concentration, Cu B 2+ is the target and the nitrite derivative (a 3 3+ NO 2 -Cu B 2+ ) is formed.…”

Section: Discussionmentioning

confidence: 96%

Control of respiration by nitric oxide in Keilin-Hartree particles, mitochondria and SH-SY5Y neuroblastoma cells

Mastronicola

Genova

Arese

et al. 2003

Cellular and Molecular Life Sciences (CMLS)

View full text Add to dashboard Cite

The pattern of cytochrome c oxidase inhibition by nitric oxide (NO) was investigated polarographically using Keilin-Hartree particles, mitochondria and human neuroblastoma cells. NO reacts with purified cytochrome c oxidase forming either a nitrosyl- or a nitrite-inhibited derivative, displaying distinct kinetics and light sensitivity of respiration recovery in the absence of free NO. Keilin-Hartree particles or cells, respiring either on endogenous substrates alone or in the presence of ascorbate, as well as state 3 and state 4 mitochondria respiring on glutamate and malate, displayed the rapid recovery characteristic of the nitrite derivative. All systems, when respiring in the presence of tetramethyl-p-phenylenediamine, were characterised by the slower, light-sensitive recovery typical of the nitrosyl derivative. Together the results suggest that the reaction of NO with cytochrome c oxidase in situ follows two alternative inhibition pathways, depending on the electron flux through the respiratory chain.

show abstract

Nitric Oxide Reacts with the Single-electron Reduced Active Site of Cytochrome c Oxidase

Cited by 32 publications

References 31 publications

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

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

Reversal of Cyanide Inhibition of Cytochrome c Oxidase by the Auxiliary Substrate Nitric Oxide

Control of respiration by nitric oxide in Keilin-Hartree particles, mitochondria and SH-SY5Y neuroblastoma cells

Contact Info

Product

Resources

About