Respiratory complex I, (also known as NADH:ubiquinone oxidoreductase, Type I NADH dehydrogenase and mitochondrial complex I) is the first large protein complex of the respiratory chains of many organisms from bacteria to humans. It catalyzes the transfer of electrons from NADH to coenzyme Q10 (CoQ10) and translocates protons across the inner mitochondrial membrane in eukaryotes or the plasma membrane of bacteria.
This enzyme is essential for the normal functioning of cells, and mutations in its subunits lead to a wide range of inherited neuromuscular and metabolic disorders. Defects in this enzyme are responsible for the development of several pathological processes such as ischemia/reperfusion damage (stroke and cardiac infarction), Parkinson's disease and others.
Function
Complex I is the first enzyme of the mitochondrial electron transport chain. There are three energy-transducing enzymes in the electron transport chain - NADH:ubiquinone oxidoreductase (complex I), Coenzyme Q – cytochrome c reductase (complex III), and cytochrome c oxidase (complex IV). Complex I is the largest and most complicated enzyme of the electron transport chain.
The reaction catalyzed by complex I is:
NADH + H+ + CoQ + 4H+in→ NAD+ + CoQH2 + 4H+out
In this process, the complex translocates four protons across the inner membrane per molecule of oxidized NADH, helping to build the electrochemical potential difference used to produce ATP. Escherichia coli complex I (NADH dehydrogenase) is capable of proton translocation in the same direction to the established Δψ, showing that in the tested conditions, the coupling ion is H+. Na+ transport in the opposite direction was observed, and although Na+ was not necessary for the catalytic or proton transport activities, its presence increased the latter. H+ was translocated by the Paracoccus denitrificans complex I, but in this case, H+ transport was not influenced by Na+, and Na+ transport was not observed. Possibly, the E. coli complex I has two energy coupling sites (one Na+ independent and the other Na+dependent), as observed for the Rhodothermus marinus complex I, whereas the coupling mechanism of the P. denitrificans enzyme is completely Na+ independent. It is also possible that another transporter catalyzes the uptake of Na+. Complex I energy transduction by proton pumping may not be exclusive to the R. marinus enzyme. The Na+/H+ antiport activity seems not to be a general property of complex I. However, the existence of Na+-translocating activity of the complex I is still in question.
The reaction can be reversed – referred to as aerobic succinate-supported NAD+ reduction by ubiquinol – in the presence of a high membrane potential, but the exact catalytic mechanism remains unknown. Driving force of this reaction is a potential across the membrane which can be maintained either by ATP-hydrolysis or by complexes III and IV during succinate oxidation.
Complex I may have a role in triggering apoptosis. In fact, there has been shown to be a correlation between mitochondrial activities and programmed cell death (PCD) during somatic embryo development.
Complex I is not homologous to Na+-translocating NADH Dehydrogenase (NDH) Family (TC# 3.D.1), a member of the Na+ transporting Mrp superfamily.
As a result of a two NADH molecule being oxidized to NAD+, three molecules of ATP can be produced by Complex V (ATP synthase) downstream in the respiratory chain.
Mechanism
Overall mechanism All redox reactions take place in the hydrophilic domain of complex I. NADH initially binds to complex I, and transfers two electrons to the flavin mononucleotide (FMN) prosthetic group of the enzyme, creating FMNH2. The electron acceptor – the isoalloxazine ring – of FMN is identical to that of FAD. The electrons are then transferred through the FMN via a series of iron-sulfur (Fe-S) clusters, and finally to coenzyme Q10 (ubiquinone). This electron flow changes the redox state of the protein, inducing conformational changes of the protein which alters the pK values of ionizable side chain, and causes four hydrogen ions to be pumped out of the mitochondrial matrix. Ubiquinone (CoQ) accepts two electrons to be reduced to ubiquinol (CoQH2).
Electron transfer mechanism The proposed pathway for electron transport prior to ubiquinone reduction is as follows: NADH – FMN – N3 – N1b – N4 – N5 – N6a – N6b – N2 – Q, where Nx is a labelling convention for iron sulfur clusters. The high reduction potential of the N2 cluster and the relative proximity of the other clusters in the chain enable efficient electron transfer over long distance in the protein (with transfer rates from NADH to N2 iron-sulfur cluster of about 100 μs).
The equilibrium dynamics of Complex I are primarily driven by the quinone redox cycle. In conditions of high proton motive force (and accordingly, a ubiquinol-concentrated pool), the enzyme runs in the reverse direction. Ubiquinol is oxidized to ubiquinone, and the resulting released protons reduce the proton motive force.
Proton translocation mechanism The coupling of proton translocation and electron transport in Complex I is currently proposed as being indirect (long range conformational changes) as opposed to direct (redox intermediates in the hydrogen pumps as in heme groups of Complexes III and IV). The architecture of the hydrophobic region of complex I shows multiple proton transporters that are mechanically interlinked. The three central components believed to contribute to this long-range conformational change event are the pH-coupled N2 iron-sulfur cluster, the quinone reduction, and the transmembrane helix subunits of the membrane arm. Transduction of conformational changes to drive the transmembrane transporters linked by a 'connecting rod' during the reduction of ubiquinone can account for two or three of the four protons pumped per NADH oxidized. The remaining proton must be pumped by direct coupling at the ubiquinone-binding site. It is proposed that direct and indirect coupling mechanisms account for the pumping of the four protons.
The N2 cluster's proximity to a nearby cysteine residue results in a conformational change upon reduction in the nearby helices, leading to small but important changes in the overall protein conformation. Further electron paramagnetic resonance studies of the electron transfer have demonstrated that most of the energy that is released during the subsequent CoQ reduction is on the final ubiquinol formation step from semiquinone, providing evidence for the "single stroke" H+ translocation mechanism (i.e. all four protons move across the membrane at the same time). Alternative theories suggest a "two stroke mechanism" where each reduction step (semiquinone and ubiquinol) results in a stroke of two protons entering the intermembrane space.
The resulting ubiquinol localized to the membrane domain interacts with negatively charged residues in the membrane arm, stabilizing conformational changes. An antiporter mechanism (Na+/H+ swap) has been proposed using evidence of conserved Asp residues in the membrane arm. The presence of Lys, Glu, and His residues enable for proton gating (a protonation followed by deprotonation event across the membrane) driven by the pKa of the residues.
Composition and structure
NADH:ubiquinone oxidoreductase is the largest of the respiratory complexes. In mammals, the enzyme contains 44 separate water-soluble peripheral membrane proteins, which are anchored to the integral membrane constituents. Of particular functional importance are the flavin prosthetic group (FMN) and eight iron-sulfur clusters (FeS). Of the 44 subunits, seven are encoded by the mitochondrial genome.
The structure is an "L" shape with a long membrane domain (with around 60 trans-membrane helices) and a hydrophilic (or peripheral) domain, which includes all the known redox centres and the NADH binding site. All thirteen of the E. coli proteins, which comprise NADH dehydrogenase I, are encoded within the nuo operon, and are homologous to mitochondrial complex I subunits. The antiporter-like subunits NuoL/M/N each contains 14 conserved transmembrane (TM) helices. Two of them are discontinuous, but subunit NuoL contains a 110 Å long amphipathic α-helix, spanning the entire length of the domain. The subunit, NuoL, is related to Na+/ H+ antiporters of TC# 2.A.63.1.1 (PhaA and PhaD).
Three of the conserved, membrane-bound subunits in NADH dehydrogenase are related to each other, and to Mrp sodium-proton antiporters. Structural analysis of two prokaryotic complexes I revealed that the three subunits each contain fourteen transmembrane helices that overlay in structural alignments: the translocation of three protons may be coordinated by a lateral helix connecting them.
Complex I contains a ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron-sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal beta-sheet of the 49-kDa subunit. All 45 subunits of the bovine NDHI have been sequenced. Each complex contains noncovalently bound FMN, coenzyme Q and several iron-sulfur centers. The bacterial NDHs have 8-9 iron-sulfur centers.
A recent study used electron paramagnetic resonance (EPR) spectra and double electron-electron resonance (DEER) to determine the path of electron transfer through the iron-sulfur complexes, which are located in the hydrophilic domain. Seven of these clusters form a chain from the flavin to the quinone binding sites; the eighth cluster is located on the other side of the flavin, and its function is unknown. The EPR and DEER results suggest an alternating or "roller-coaster" potential energy profile for the electron transfer between the active sites and along the iron-sulfur clusters, which can optimize the rate of electron travel and allow efficient energy conversion in complex I.