Nitric oxide and peroxynitrite cause irreversible increases in the Km for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies

Abstract

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K(m) for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K(m). These include: treatment of the purified enzyme with peroxynitrite or high (microM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite

“…O 2 ⅐ Ϫ generated from that location is released both into the matrix and intermembrane space (51), where it is dismutated to hydrogen peroxide (H 2 O 2 ) by manganese superoxide dismutase (MnSOD) and copper zinc SOD (CuZnSOD), respectively (29). Hence, mitochondrial NO metabolism involves regulation of O 2 consumption, H 2 O 2 production, and the effects of the freely diffusible (to the cytosol) H 2 O 2 on cell signaling and gene expression (11).NO and O 2 ⅐ Ϫ combine at a diffusion-limited reaction rate to form peroxynitrite (ONOO Ϫ ) (40), which is able to nitrate and/or oxidize amino acid side chains of mitochondrial proteins and result in irreversible inhibition of NADH-ubiquinone reductase (complex I), succinate-ubiquinone reductase (complex II), complex IV, aconitase, and ATPase (12,13,18,65). In agreement with its effects on ETC components, ONOO Ϫ potentiated the inhibition of respiration in isolated cardiac muscle compared with the inhibition elicited by NO alone and made it irreversible with time (79).…”

“…NO and O 2 ⅐ Ϫ combine at a diffusion-limited reaction rate to form peroxynitrite (ONOO Ϫ ) (40), which is able to nitrate and/or oxidize amino acid side chains of mitochondrial proteins and result in irreversible inhibition of NADH-ubiquinone reductase (complex I), succinate-ubiquinone reductase (complex II), complex IV, aconitase, and ATPase (12,13,18,65). In agreement with its effects on ETC components, ONOO Ϫ potentiated the inhibition of respiration in isolated cardiac muscle compared with the inhibition elicited by NO alone and made it irreversible with time (79).…”

Nitric oxide irreversibly inhibits cytochrome oxidase at low oxygen concentrations: Evidence for inverse oxygen concentration‐dependent peroxynitrite formation

“…Figures 1A-1C show that NO added to purified COX causes irreversible COX inhibition. Peroxynitrite is a powerful oxidative NO congener that irreversibly inhibits the activity of various mitochondrial proteins including COX (7,17). Uric acid is a well-known peroxynitrite scavenger (17) that protects against peroxynitrite-induced damaging mitochondrial components (10,18).…”

Competitive, Reversible, Physiological? Inhibition of Mitochondrial Cytochrome Oxidase by Nitric Oxide

“…Cytochrome oxidase damage can also be induced by peroxynitrite (41,42), making it unclear in the cell whether it is NO itself that is acting directly on the enzyme (although in the case of the purified enzyme NO can be shown to be acting directly). We have recently shown that the irreversible damage caused by NO and peroxynitrite primarily exhibits itself as a raising of the K m for oxygen (38). The oxygen K m is normally so low (submicromolar), and the majority of in vitro assays for cytochrome oxidase damage are performed at such high oxygen tensions (240 mM), that small increases in this K m are not detectable; this results in an underestimation of the extent of damage to the enzyme.…”

“…However, higher levels of NO (38), and/or, prolonged exposure in cell culture (39) results in significant irreversible damage. Neurones seem particularly susceptible to these irreversible effects (40).…”