Complex I Nadh Ubiquinone Oxidoreductase

During oxidative phosphorylation, electrons pass through a series of membrane-bound multiprotein complexes that translocate protons across the membrane, resulting in a proton-motive force used by ATP synthase to make ATP (1). It couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the inner mitochondrial membrane. Complex I is the first of these electron transfer complexes and accounts for up to 40% of the pumped protons (50). In human mitochondria, complex I is also a major source of activated 02 species. Complex I or the NADH-ubiquinone oxidoreductase is a macromolecular assembly of 43 subunits catalyzing electron transfer from NADH to ubiquinone through flavin mononucleotide and up to 7 iron-sulfur clusters (107). It is one of the most complex subunits known, with a molecular mass of about 1000 kDa (80). Thirty six of complex I's subunits are encoded by nuclear DNA whilst 7 are encoded by mitochondrial DNA. This includes several prosthetic groups including FMN, at least 7 Fe-S clusters, and five molecules of protein bound coenzyme Q; a natural acceptor of electrons. Complex I is the first site of oxidative phosphorylation but its function is still incompletely understood (21). It is hypothesized that Fe-S clusters might act as redox centers; however, the precise location within the cluster is still elusive.

Early experiments proved the involvement ofcomplex I in AOS production. The addition of low concentrations of either NADH or NADPH, which feeds the electron to complex I at a slow rate, leads to copious AOS production, detected by lipid peroxidation (118). In another study, water-soluble CoQ homologues used as an electron acceptor from isolated complex I stimulated H202 production, which in turn was partly inhibited by rotenone, indicating that water-soluble quinones may react with 02 when reduced at sites both prior and subsequent to the rotenone block (108). Complex I is very sensitive to a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. Affinity labeling of complex I by rotenone analogues has shown recently that a 23 kDa PSST subunit of complex I is the likely binding site for these inhibitors and thus is the subunit that plays a key role in electron transfer by functionally coupling iron-sulfur cluster to quinone (108). The proximity ofthis binding site to a possible 4 Fe-S cluster in PSST suggests that this subunit is the site of final transfer of electrons, the Fe-S proteins to quinone and a likely site for semiquinone generation. There is evidence that the one-electron donor to oxygen in complex I is a non-physiological quinone reduction site. The cDNA of the human PSST protein was used to investigate tissue-specific expression and to localize the gene for this subunit to chromosome 19pl3 (45). It is a hydrophilic site, which reduces several quinones to semiquinone, and since they are unstable, can reduce 02 to 02'~ (30). Thus, it may represent a potential site for 02" generation. Indeed, duroquinone, a soluble analog of co-enzyme Q, constricts PAs and inhibits IK in PASMCs (99).

In addition to direct studies for 02" generation measurements, complex I deficiency (and its associated diseases) has been shown to generate AOS. Complex I deficiency displays a wide spectrum of phenotypes ranging from exercise intolerance to cataracts and development delay, which includes Leigh disease characterized by degeneration of basal ganglia and/or cardiomyopathy with or without cataract, and fatal infantile lactic acidosis (101). A more detailed investigation showed the correlation of increased levels of expression ofMnSOD (a compensatory response) with a poor prognosis in complex I deficiency (91, 101). Excess AOS production is further complicated by damage to other systems such as mitochondrial aconitase, complex I, and succinate dehydrogenase which have functional Fe-S centers (site of 02' reactivity) (34). This damage is pronounced in MnSOD knockout mice where 02'~ produced in the mitochondria is not removed by the normal radical scavenger mechanisms (56).

4.2. Complex II and IV

Complex II is a flavoprotein dehydrogenase which uses succinate as an exogenous substrate. The main function of this complex is to transfer the electrons from FADH2 (reduced ubiquinone) to FAD+ (oxidized ubiquinol). Complex II is generally involved in electron flux from complex I to complex III. Similarly, complex IV, cytochrome oxidase, is composed of two cytochromes, a and a3, and accepts electrons from reduced cytochrome c and passes them to

02. Thus, complexes II and IV are involved in shuttling electrons but play no role in generation of AOS. Inhibition of complex IV with cyanide does cause a transient pulmonary vasoconstriction, associated with increased AOS production (10). Recently we have demonstrated thatwhile A^ does depolarize in response to cyanide in 02-sensitive SMCs, these mitochondria are much less sensitive than those in cardiac cell lines, consistent with there being functionally different mitochondria in different cardiovascular tissues (72, 74).

4.3. Complex III (Ubiquinol-cytochrome c Oxidoreductase)

Complex III is responsible for taking reducing equivalents from complex II contained in ubiquinol and transferring them through reactions with cytochrome b, the Rieske Fe-S protein and cytochrome c„ to the final electron acceptor cytochrome c. In the process, two species of semiquinone are generated. The Q-cycle mechanism proposed for the operation of the ubiquinol cytochrome c reductase outlines these two sites. Ubiquinol donates one electron to the Rieske Fe-S protein (a myxothiazol-inhibitor site) generating a semiquinone in proximity to the outer face of the inner membrane, which then reduces the first cytochrome b heme (bL) (121). The second cytochrome b heme (bH), situated closer to the matrix side of the membrane, accepts an electron from the first heme (bL) and reduces ubiquinone to form ubisemiquinone and, with the passage of another electron, ubiquinol (the antimycin A-inhibitor site) (121). The transfer of the first electron is a relative low potential transfer with a redox potential of about +200 mV, however, the loss of the second electron from the resulting semiquinone anion to fully oxidized ubiquinol is much more energetic with a redox potential of about -200 mV. Thus, two ubiquinone molecules are oxidized at the outer site, transferring two electrons to cytochrome c (77).

The two inhibitors antimycin and myxothiazol, although they inhibit complex III electron transfer equally, have dramatically different effects on 02'~ production. Blocking electron passage out of cytochrome bH prevents the semiquinone from donating its electron and so, inhibition with antimycin, in some models, produces a more than tenfold increase in 02'~ formation because it prevents ubisemiquinone formation at the cytosolic side of the inner mitochondrial membrane (57). However, in the pulmonary circulation we have found that antimycin, like rotenone, reduces normoxic generation of AOS (10, 19). Whether this reflects measurement errors or true differences in the effect of the inhibitors that relate to the type of mitochondria under study is unknown. Recent data suggest that there are significant differences in mitochondrial respiration and AOS production between lung and renal mitochondria (72).

However, the question remains about the relative importance and contribution of complexes I and III in 02 sensing. First, let us consider the hemodynamic effects of ETC inhibitors (Fig. 6). Rotenone and antimycin, but not cyanide, cause an increase in PVR in isolated lungs, and a fall in renal vascular resistance, mimicking the effects of hypoxia. These opposing effects are also seen in isolated PA vs. RA rings (72). In the first systematic exploration of the theory that mitochondria are the pulmonary vascular 02 sensors we showed that inhibiting complexes I and III (but not complex IV) caused PA constriction, inhibited subsequent HPV and like hypoxia reduced levels of AOS.

Nadh Ubiquinone Oxidoreductase

Figure 6\ The effects of ETC inhibitors and hypoxia on AOS generation and PVR are concordant. In the Krebs'-albumin perfused isolated rat lungs, PA pressure reflects pulmonary vascular resistance because flow is held constant. Chemiluminescence, measured by two different methods (luminol and lucigenin enhancement), is decreased (open bars) whereas PA pressure is increased (solid bars) in response to rotenone and antimycin (Reproduced from Ref. 10).

Figure 6\ The effects of ETC inhibitors and hypoxia on AOS generation and PVR are concordant. In the Krebs'-albumin perfused isolated rat lungs, PA pressure reflects pulmonary vascular resistance because flow is held constant. Chemiluminescence, measured by two different methods (luminol and lucigenin enhancement), is decreased (open bars) whereas PA pressure is increased (solid bars) in response to rotenone and antimycin (Reproduced from Ref. 10).

Leach et al. reported that inhibition of complex I with rotenone resulted in inhibition of HPV which could not be reversed by bypassing complex I by providing succinate as a substrate for complex II (53). They found that succinate successfully reversed the rotenone-induced inhibition HPV. Moreover, inhibition of complex III abolished HPV. They concluded that complex III is the primary site for AOS production. However, studies published by our laboratory show otherwise (72, 74). Both rotenone and antimycin cause PA constriction of a similar magnitude to that elicited by hypoxia. Furthermore, these agents inhibit subsequent HPV (10). It is also noteworthy that chronic hypoxia decreases the constriction to both hypoxia and rotenone, while the pressor response to other vasoconstrictors is enhanced, suggesting again that hypoxia and certain ETC inhibitors share a common pathway (97). Cyanide does not mimic hypoxia in our hands (10), nor does it do so in studies by another group (125). We have also found that rotenone and antimycin, like hypoxia, decrease H202 levels regardless of the type of assays used (72). Thus, it provides evidence that perhaps both complexes I and III contribute to the AOS pool (72). Furthermore, a similar observation was made in DA where use of rotenone and antimycin A separately and in combination successfully relaxed 02-preconstricted DA (74). In addition, these observations have been extended to the level of 02-sensitive K+ channel modulation and regulation of PA and DA tone. Thus, recent evidence suggests that both complexes I and III contribute to the AOS pool as suggested by our laboratory a decade ago. The difference in the conclusion reached by Leach et al. (53) and our laboratory might be due to the presence of PGF2 and use of myxothiazol, a more proximal complex III inhibitor, in the perfusion system.

Moreover, use of rotenone in conjunction with succinate has been shown to generate reversed electron transport and methodological artifacts (114). Waypa's conclusion that the mitochondria function as 02 sensors during hypoxia and that ROS generated in the proximal ETC act as second messengers to trigger calcium increases in PASMCs during acute hypoxia (125) mirrors our earlier conclusions (7, 10). However, our groups differ in two regards. We find AOS reduced in acute hypoxia (9, 13, 14, 97, 98) and we have evidence that complex I, as well as complex III, participate in the mechanism of HPV and 02 responses in the human ductus arteriosus (74).

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  • Azzurra
    What complex is reduced ubiquinone?
    8 years ago

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