Pumping protons: a complex problem.
Cells use a lot of ATP. An E.coli cell can use a billion ATP molecules per minute and humans use their own weight in ATP every day. Because of this, cells need an efficient mechanism to recombine ADP with phosphate to regenerate ATP, and much of the energy in our food is used in this process. ATP synthase, the enzyme that makes ATP, is located in the mitochondrial inner membrane (or the bacterial cell membrane), and is powered by a concentration difference (gradient) of protons across the membrane. Four different protein complexes make up the respiratory chain that pumps protons across the cell membrane: ubiquinone oxidoreductase (complex I), succinate dehydrogenase (complex II), cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV). These generate the proton gradient that drives ATP synthase (Figure 1). Complex I oxidises NADH (formed in glycolysis and the Krebs cycle) to pump four protons across the membrane. Complex I also uses the reductive power of NADH to reduce quinone, which is then used as a substrate by complex III to pump two more protons across the membrane.
One ‘ell of a structure.
In mammals, complex I consists of 44 different subunits and electron microscopy studies have revealed that it has an L shape. One arm of the L is inserted in the membrane (the membrane arm) and the other arm (the hydrophilic arm) projects some 100 Å into the mitochondrial matrix. Bacteria also possess a complex I, but it is a ‘cut down’ version of its eukaryotic counterpart, containing at least 14 of the subunits found in the mitochondrial enzyme. The crystal structure of complex I from the bacterium Thermus thermophilus (PDB entry 4hea) shows that it also has the characteristic L shape (view-1) with the hydrophilic arm projecting into the cytoplasm.
The complex consists of 16 protein subunits with a combined molecular weight of 536 . The complex contains seven Fe4S4 and two Fe2S2 iron-sulfur clusters and a bound flavin mononucleotide (FMN) as (Figure 2). Seven of the subunits span the membrane and contain 64 transmembrane helices in total.
Where does NADH enter complex I?
The NADH substrate-binding site is at the distal end of the hydrophilic arm in subunit Nqo1 (view-2). The site is formed from a modified which also incorporates a FMN-binding site. The structure of the hydrophilic arm of T. thermophilus complex I with bound NADH (PDB entry 3iam) shed light on the interactions between FMN and NADH. The adenine ring of NADH is held in place by stacking against three phenylalanine residues. This allows the nicotinamide ring of NADH to stack against the isolloxazine ring of the bound FMN (view-2). Electron transfer then occurs between the C4N atom of the nicotinamide ring of NADH and N5 of FMN. The architecture of the binding site positions these atoms within 3.2 Å of each other for efficient electron transfer. A glutamate residue in the binding site appears to help position the two rings close together.
Reducing the problem.
Subunits Nqo1, Nqo3, Nqo6 and Nqo9 contain seven of the iron-sulfur clusters in the complex. These form a “wire” some 90 Å long which carries electrons from the FMN, through the hydrophilic arm to the quinone-binding site formed by Nqo4, Nqo6 and Nqo8 (view-3). The iron-sulfur clusters are at most 14 Å apart (edge to edge distance), allowing efficient electron transfer between the clusters (view-3). Nqo2 and Nqo3 contain additional iron-sulfur clusters, but the function of these clusters is unknown.
NADH donates two electrons to the iron-sulfur cluster “wire” which are transferred one at a time along the clusters and used to reduce the bound quinone. Different organisms use different quinones, and T. thermophilus uses menaquinone-8 (Figure 3). Menaquinone-8 binds in a 30 Å long cavity formed by subunits Nqo4, Nqo6 and Nqo8 at the interface between the two arms. The entrance to the cavity, through which the quinone has to pass, is surprisingly narrow, around 2.3 x 4.5 Å (view-4), suggesting movement in this region to allow quinone binding. The quinone-binding cavity contains hydrophilic residues along its length, which may interact with the hydrophilic head group of the quinone as it enters the cavity. Residues His38, Tyr87 and Asp139 in Nqo4, at the distal end of the cavity, probably position the quinone close to the last iron-sulfur cluster in the “wire” to allow for efficient reduction of the quinone. After being reduced, the quinone leaves complex I and is subsequently used as a substrate by complex III.
We all pump together.
When complex I reduces the bound quinone it also pumps four protons across the membrane. Complex I contains four potential channels within the membrane which are likely to perform this role. Subunits Nqo12, 13 and 14 are homologous to each other and to sodium/proton antiporters, indicating that they most likely form the proton-pumping channels 1, 2 and 3, respectively. Subunits Nqo8, Nqo10 and Nqo11 also form a channel across the membrane, channel 4, which links the other channels to the hydrophilic domain (view-5).
So how does reduction of the quinone result in the pumping of four protons across the membrane? The most obvious assumption is that each channel pumps a single proton. But this begs the question how the channels are coordinated, especially as the distal channel is 130 Å from the quinone binding site. Each channel contains two transmembrane helices which have a break in the middle with a charged residue at this point (view-5). It is likely that these charged residues form a path for the proton as it makes its way through the channel. Other charged residues appear to connect the proton pumping channels together, possibly providing a mechanism of coordination between the channels. The C-terminus of Nqo12 (containing channel 1) consists of a 104 Å long α-helix which runs along the surface of the membrane before terminating adjacent to Nqo14. The role of this helix is unknown, although it interacts with one of the two broken transmembrane helices in each of the channels. Additional charged residues link the quinone binding site to the adjacent channel (channel 4), suggesting that they may initiate proton pumping upon reduction of the quinone (view-5). Further work is needed to investigate the mechanism of proton pumping.
A long-distance relationship that works.
NADH is used as a reducing agent by complex I, donating two electrons that reduce a quinone molecule and drive the pumping of four protons across the membrane in four channels. Amazingly, the NADH substrate is 85 Å away from the quinone and the quinone is 140 Å removed from the most distant channel! Complex I displays a truly remarkable form of long-distance communication.
AcknowledgementThis Quips article was developed in collaboration with Leo Sazanov at the MRC Mitochondrial Biology Unit, Cambridge.