Redox-induced activation of the proton pump in the respiratory complex I.

Complex I functions as a redox-linked proton pump in the respiratory chains of mitochondria and bacteria, driven by the reduction of quinone (Q) by NADH. Remarkably, the distance between the Q reduction site and the most distant proton channels extends nearly 200 Å. To elucidate the molecular origin of this long-range coupling, we apply a combination of large-scale molecular simulations and a site-directed mutagenesis experiment of a key residue. In hybrid quantum mechanics/molecular mechanics simulations, we observe that reduction of Q is coupled to its local protonation by the His-38/Asp-139 ion pair and Tyr-87 of subunit Nqo4. Atomistic classical molecular dynamics simulations further suggest that formation of quinol (QH2) triggers rapid dissociation of the anionic Asp-139 toward the membrane domain that couples to conformational changes in a network of conserved charged residues. Site-directed mutagenesis data confirm the importance of Asp-139; upon mutation to asparagine the Q reductase activity is inhibited by 75%. The current results, together with earlier biochemical data, suggest that the proton pumping in complex I is activated by a unique combination of electrostatic and conformational transitions.

Real-time optical studies of respiratory Complex I turnover.

Reduction of Complex l (NADH:ubiquinone oxidoreductase l) from Escherichia coli by NADH was investigated optically by means of an ultrafast stopped-flow approach. A locally designed microfluidic stopped-flow apparatus with a low volume (0.21Jl) but a long optical path (10 mm) cuvette allowed measurements in the time range from 270 ).IS to seconds. The data acquisition system collected spectra in the visible range every 50 )JS. Analysis of the obtained time-resolved spectral changes upon the reaction of Complex I with NADH revealed three kinetic components with characteristic times of <270 ).IS, 0.45-0.9 ms and 3-6 ms, reflecting reduction of different FeS clusters and FMN. The rate of the major ( T = 0.45-0.9 ms) component was slower than predicted by electron transfer theory for the reduction of all FeS clusters in the intraprotein redox chain. This delay of the reaction was explained by retention of NAD+ in the catalytic site. The fast optical changes in the time range of 0.27- 1.5 ms were not altered significantly in the presence of 1 0-fold excess of NAD+ over NADH. The data obtained on the NuoF E95Q variant of Complex I shows that the single amino acid replacement in the catalytic site caused a strong decrease of NADH binding and/or the hydride transfer from bound NADH to FMN.

A single amino acid residue controls ROS production in the respiratory Complex I from Escherichia coli.

Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)-binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O2 ). In the E95Q variant short-chain ubiquinones exhibit strong artificial one-electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS-generating state. We propose the existence of two (closed and open) states of the NAD(+) -bound enzyme as one feature of the substrate-binding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild-type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O2 in contrast to E95Q variant.

Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis.

The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed.

High affinity cation-binding sites in Complex I from Escherichia coli.

Studies on the activity of Complex I from Escherichia coli in the presence of different metal cations revealed at least two high affinity metal-binding sites. Membrane-bound or isolated Complex I was activated by K(+) (apparent binding constant approximately 125 microM) and inhibited by La(3+) (IC(50)= 1 microM). K(+) and La(3+) do not occupy the same site. Possible localization of these metal-binding sites and their implication in catalysis are discussed.

The role of the invariant glutamate 95 in the catalytic site of Complex I from Escherichia coli.

Replacement of glutamate 95 for glutamine in the NADH- and FMN-binding NuoF subunit of E. coli Complex I decreased NADH oxidation activity 2.5-4.8 times depending on the used electron acceptor. The apparent K(m) for NADH was 5.2 and 10.4 microM for the mutant and wild type, respectively. Analysis of the inhibitory effect of NAD(+) on activity showed that the E95Q mutation caused a 2.4-fold decrease of K(i)(NAD+) in comparison to the wild type enzyme. ADP-ribose, which differs from NAD(+) by the absence of the positively charged nicotinamide moiety, is also a competitive inhibitor of NADH binding. The mutation caused a 7.5-fold decrease of K(i)(ADP-ribose) relative to wild type enzyme. Based on these findings we propose that the negative charge of Glu95 accelerates turnover of Complex I by electrostatic interaction with the negatively charged phosphate groups of the substrate nucleotide during operation, which facilitates release of the product NAD(+). The E95Q mutation was also found to cause a positive shift of the midpoint redox potential of the FMN, from -350 mV to -310 mV, which suggests that the negative charge of Glu95 is also involved in decreasing the midpoint potential of the primary electron acceptor of Complex I.

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I).

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.

Ca2+ stabilization of respiratory complex I from Escherichia coli.

Stability of the membrane-bound and purified H+-translocating NADH:ubiquinone oxidoreductase, Complex I, was studied. The loss of the enzyme activity is strongly increased by alkaline pH and dilution of the sample. Complex I inactivation is prevented specifically by a low concentration of Ca2+ and/or an intracellular stabilization factor (ISF). The action of both, Ca2+ and ISF, on Complex I stability is interdependent. The data are discussed in terms of a release of structural Ca2+ as a reason for Complex I decay and an effect of ISF on the affinity and/or accessibility of Ca2+-binding site.

Activation of respiratory Complex I from Escherichia coli studied by fluorescent probes.

Respiratory Complex I from E. coli may exist in two interconverting forms: resting (R) and active (A). The R/A transition of purified, solubilized Complex I occurring upon turnover was studied employing two different fluorescent probes, Annine 6+, and NDB-acetogenin. NADH-induced fluorescent changes of both dyes bound to solubilized Complex I from E. coli were characterized as a function of the protein:dye ratio, temperature, ubiquinone redox state and the enzyme activity. Analysis of this data combined with time-resolved optical measurements of Complex I activity and spectral changes indicated two ubiquinone-binding sites; a possibility of reduction of the tightly-bound quinone in the resting state and reduction of the loosely-bound quinone in the active state is discussed. The results also indicate that upon the activation Complex I undergoes conformational changes which can be mapped to the junction of the hydrophilic and membrane domains in the region of the assumed acetogenin-binding site.

Role of the conserved arginine 274 and histidine 224 and 228 residues in the NuoCD subunit of complex I from Escherichia coli.

The conserved arginine 274 and histidine 224 and 228 residues in subunit NuoCD of complex I from Escherichia coli were substituted for alanine. The wild-type and mutated NuoCD subunit was expressed on a plasmid in an E. coli strain bearing a nuoCD deletion. Complex I was fully expressed in the H224A and H228A mutants, whereas the R274A mutation yielded approximately 50% expression. Ubiquinone reductase activity of complex I was studied in membranes and with purified enzyme and was 50% and 30% of the wild-type activity in the H224A and H228A mutants, respectively. The activity of R274A was less than 5% of the wild type in membranes but 20% in purified complex I. Rolliniastatin inhibited quinone reductase activity in the mutants with similar affinity as in the wild type, indicating that the quinone-binding site was not significantly altered by the mutations. Ubiquinone-dependent superoxide production by complex I was similar to the wild type in the R274A mutant but slightly higher in the H224A and H228A mutants. The EPR spectra of purified complex I from the H224A and H228A mutants did not differ from the wild type. In contrast, the signals of the N2 cluster and another fast-relaxing [4Fe-4S] cluster, tentatively assigned as N6b, were drastically decreased in the NADH-reduced R274A mutant enzyme but reappeared on further reduction with dithionite. These findings show that the redox potential of the N2 and N6b centers is shifted to more negative values by the R274A mutation. Purified complex I was reconstituted into liposomes, and electric potential was generated across the membrane upon NADH addition in all three mutant enzymes, suggesting that none of the mutations directly affect the proton-pumping machinery.

Regulation of expression of Na+ -translocating NADH:quinone oxidoreductase genes in Vibrio harveyi and Klebsiella pneumoniae.

The expression of genes encoding sodium-translocating NADH:quinone oxidoreductase (Na(+)-NQR) was studied in the marine bacterium Vibrio harveyi and in the enterobacterium Klebsiella pneumoniae. It has been shown that such parameters as NaCl concentration, pH value, and presence of an uncoupler in the growth media do not influence significantly the level of nqr expression. However, nqr expression depends on the growth substrates used by these bacteria. Na(+)-NQR is highly repressed in V. harveyi during anaerobic growth, and nqr expression is modulated by electron acceptors and values of their redox potentials. The latter effect was shown to be independent of the ArcAB regulatory system.