Electron and proton transfer in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

Cells of every living organism on our planet − bacterium, plant or animal − are organized in such a way that despite differences in structure and function they utilize the same metabolic energy represented by electrochemical proton gradient across a membrane. This gradient of protons is generated by the series of membrane bound multisubunit proteins, Complex I, II, III and IV, organized in so-called respiratory or electron transport chain. In the eukaryotic cell it locates in the inner mitochondrial membrane while in the bacterial cell it locates in the cytoplasmic membrane. The function of the respiratory chain is to accept electrons from NADH and ubiquinol and transfer them to oxygen resulting in the formation of water. The free energy released upon these redox reactions is converted by respiratory enzymes into an electrochemical proton gradient, which is used for synthesis of ATP as well as for many other energy dependent processes. This thesis is focused on studies of the first member of the respiratory chain − NADH:ubiquinone oxidoreductase or Complex I. This enzyme has a boot-shape structure with hydrophilic and hydrophobic domains, the former of which has all redox groups of the protein, the flavin and eight to nine iron-sulfur clusters. Complex I serves as a proton pump coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. So far the mechanism of energy transduction by Complex I is unknown. In the present study we applied a set of different methods to study the electron and proton transfer reactions in Complex I from Escherichia coli. The main achievement was the experiment that showed that the electron transfer through the hydrophilic domain of Complex I is unlikely to be coupled to proton transfer directly or to conformational changes in the protein. In this work for the first time properties of all redox centers of Complex I were characterized in the intact purified bacterial enzyme. We also probed the role of several conserved amino acid residues in the electron transfer of Complex I. Finally, we found that highly conserved amino acid residues in several membrane subunits form a common pattern with a very prominent feature – the presence of a few lysines within the membrane. Based on the experimental data, we suggested a tentative principle which may govern the redox-coupled proton pumping in Complex I.

Role of lipid peroxidation in impairment of mitochondrial function at complex I by methyl isocyanate treatment of rats in vivo

The subcutaneous administration of methyl isocyanate (MIC) in 1.0 LD50 dose in rats caused a significant effect on hepatic mitochondrial function only at complex I region of the respiratory chain. MIC administration at 1.0 LD50 dose also resulted in significant increases in malondialdehyde and ferrous ion concentration in liver mitochondria. It is suggested that the augmented lipid peroxidation in hepatic mitochondria, catalyzed by iron, possibly mobilized from intracellular stores leads to the inhibition of enzymes of mitochondrial respiration at complex I region, in vivo, in rats receiving a lethal dose of MIC subcutaneously.

Isolation, characterization and electron microscopic single particle analysis of the NADH : ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degreesC. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degreesC. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degreesC, with a half-life of about 10 h at 80 degreesC. The activity shows a linear Arrhenius plot at 50-85 degreesC with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90degrees) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.

Lack of Oxygen Deactivates Mitochondrial Complex I IMPLICATIONS FOR ISCHEMIC INJURY?

For S-nitrosothiols and peroxynitrite to interfere with the activity of mitochondrial complex I, prior transition of the enzyme from its active (A) to its deactive, dormant (D) state is necessary. We now demonstrate accumulation of the D-form of complex I in human epithelial kidney cells after prolonged hypoxia. Upon reoxygenation after hypoxia there was an initial delay in the return of the respiration rate to normal. This was due to the accumulation of the D-form and its slow, substrate-dependent reconversion to the A-form. Reconversion to the A-form could be prevented by prolonged incubation with endogenously generated NO. We propose that the hypoxic transition from the A-form to the D-form of complex I may be protective, because it would act to reduce the electron burst and the formation of free radicals during reoxygenation. However, this may become an early pathophysiological event when NO-dependent formation of S-nitrosothiols or peroxynitrite structurally modifies complex I in its D-form and impedes its return to the active state. These observations provide a mechanism to account for the severe cell injury that follows hypoxia and reoxygenation when accompanied by NO generation.

MEASUREMENT OF SUPEROXIDE FORMATION BY MITOCHONDRIAL COMPLEX I OF YARROWIA LIPOLYTICA

Complex I (NADH: ubiquinone oxidoreductase) is generally regarded as one of the major sources of mitochondrial reactive oxygen species (ROS). Mitochondrial membranes from the obligate aerobic yeast Yarrowia lipolytica, as well as the purified and reconstituted enzyme, can be used to measure complex I-dependent generation of superoxide (O-2(center dot-)). The use of isolated complex I excludes interference with other respiratory chain complexes and matrix enzymes during superoxide dismutase-sensitive reduction of acetylated cytochrome c. Alternately. hydrogen peroxide formation can be measured by the Amplex Red/horseradish peroxidase assay. Both methods allow the determination of complex I-generated ROS, depending on substrates (NADH, artificial ubiquinones), membrane potential, and active/deactive transition. ROS production by Yorrowia complex I in the

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I

Mitochondrial complex I (NADH: ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide: oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

S-nitrosation of mitochondrial complex I depends on its structural conformation

Nitric oxide is known to cause persistent inhibition of mitochondrial respiration as a result of S-nitrosation of NADH: ubiquinone oxidoreductase (complex I) (Clementi, E., Brown, G. C., Feelisch, M., and Moncada, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7631-7636). Little is known about whether such nitrosation occurs in physiological conditions and, if so, what are the possible cellular mechanisms. We have now found that the conformational state (active/deactive transition (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185)) of mitochondrial complex I is an important factor for the interaction of the enzyme with nitrosothiols and peroxynitrite. Only the deactivated, idle form of complex I was susceptible to inhibition by nitrosothiols and peroxynitrite. In contrast, the active form of the enzyme was insensitive to such treatment. Neither form of complex I was inhibited by nitric oxide itself. Our data suggest that the process of active/deactive transition plays an important role in the regulation of complex I activity and cellular respiration by nitric oxide. The implications of this finding for hypoxic or pathophysiological conditions in vivo are discussed.

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H(->+)/2e(-) indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H(->+)/2e(-). This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme. (c) 2006 Elsevier B.V. All rights reserved.

The redox-Bohr group associated with iron-sulfur cluster N2 of complex I

Proton pumping respiratory complex I (NADH: ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.

Proton pumping by complex I (NADH : ubiquinone oxidoreductase) from Yarrowia lipolytica reconstituted into proteoliposomes

The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex 1) is Still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of Delta pH and Delta psi. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization or the phospholipids with n-octyl-beta-D-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force, Delta pH and Delta psi, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex 1, we did not find indications for primary or secondary Na+ translocation by Y lipolytica complex I. (c) 2005 Elsevier B.V. All rights reserved.

Superoxide radical formation by pure complex I (NADH : Ubiquinone oxidoreductase) from Yarrowia lipolytica

Generation of reactive oxygen species (ROS) is increasingly recognized as an important cellular process involved in numerous physiological and pathophysiological processes. Complex I ( NADH: ubiquinone oxidoreductase) is considered as one of the major sources of ROS within mitochondria. Yet, the exact site and mechanism of superoxide production by this large membrane-bound multiprotein complex has remained controversial. Here we show that isolated complex 1 from Yarrowia lipolytica forms superoxide at a rate of 0.15% of the rate measured for catalytic turnover. Superoxide production is not inhibited by ubiquinone analogous inhibitors. Because mutant complex I lacking a detectable iron-sulfur cluster N2 exhibited the same rate of ROS production, this terminal redox center could be excluded as a source of electrons. From the effect of different ubiquinone derivatives and pH on this side reaction of complex I we concluded that oxygen accepts electrons from FMNH2 or FMN semiquinone either directly or via more hydrophilic ubiquinone derivatives.

Conformation-specific crosslinking of mitochondrial complex I

Complex I is the only component of the eukaryotic respiratory chain of which no high-resolution structure is yet available. A notable feature of mitochondrial complex I is the so-called active/de-active conformational transition of the idle enzyme from the active (A) to the de-active, (D) form. Using an amine- and sulfhydryl-reactive crosslinker of 6.8 Å length (SPDP) we found that in the D-form of complex I the ND3 subunit crosslinked to the 39 kDa (NDUFA9) subunit. These proteins could not be crosslinked in the A-form. Most likely, both subunits are closely located in the critical junction region connecting the peripheral hydrophilic domain to the membrane arm of the enzyme where the entrance path for substrate ubiquinone is and where energy transduction takes place.

Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS.

Results: We found that in the heart, t½ of complex I deactivation during ischemia was 10?min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis.

Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting.

Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

Molecular mechanism and physiological role of active-deactive transition of mitochondrial complex I

The unique feature ofmitochondrial complex I is the so-called A/D transition (active-deactive transition). The A-form catalyses rapid oxidation of NADH by ubiquinone (k ~10 min) and spontaneously converts into the D-form if the enzyme is idle at physiological temperatures. Such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. The D-form can undergo reactivation given both NADH and ubiquinone availability during slow (k ~1-10 min) catalytic turnover(s). We examined known conformational differences between the two forms and suggested a mechanism exerting A/D transition of the enzyme. In addition, we discuss the physiological role of maintaining the enzyme in the D-form during the ischaemic period. Accumulation of the D-form of the enzyme would prevent reverse electron transfer from ubiquinol to FMN which could lead to superoxide anion generation. Deactivation would also decrease the initial burst of respiration after oxygen reintroduction. Therefore the A/D transition could be an intrinsic protective mechanism for lessening oxidative damage during the early phase of reoxygenation. Exposure of Cys of mitochondrially encoded subunit ND3 makes the Dform susceptible for modification by reactive oxygen species and nitric oxide metabolites which arrests the reactivation of the D-form and inhibits the enzyme. The nature of thiol modification defines deactivation reversibility, the reactivation timescale, the status of mitochondrial bioenergetics and therefore the degree of recovery of the ischaemic tissues after reoxygenation.