Cytochrome proton translocation

Why has nature chosen this rather convoluted path for electrons in Complex 111 First of all. Complex 111 takes up two protons on the matrix side of the inner membrane and releases four protons on the cytoplasmic side for each pair of electrons that passes through the Q cycle. The apparent imbalance of two protons in ior four protons out is offset by proton translocations in Complex rV, the cytochrome oxidase complex. The other significant feature of this mechanism is that it offers a convenient way for a two-electron carrier, UQHg, to interact with the bj and bfj hemes, the Rieske protein Fe-S cluster, and cytochrome C, all of which are one-electron carriers. 

Figure 7. Mechanism of the proton-translocating ubiquinol cytochrome c reductase (complex III) Q cycle. There is a potential difference of up to 150 mV across the hydrophobic core of this complex (potential barrier represented by the vertical broken line). Cytochromes hour and b N are heme groups on the same peptide subunits of complex III which can transfer electrons across the hydrophobic core. The movement of two electrons provides the driving force to transfer two protons from the matrix to the cytosol. Diffusion of UQ and UQHj, which are uncharged, in the hydrophobic core, and lipid bilayer of the inner membrane is not influenced by the membrane potential (see Nicholls and Ferguson, 1992).

Figure 12-8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a protonpump. Q, ubiquinone C, cytochrome c F Fq, protein subunits which utilize energy from the proton gradient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H" across the membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction of H" through Fq.

The flow of electrons occurs in a similar manner from the excited pigment to cytochromes, quinones, pheophytins, ferridoxins, etc. The ATP synthase in the mitochondria of a bacterial system resembles that of the chloroplast—chloroplast proton translocating ATP synthase [37]. 

The proton-motive Q-cycle model, put forward by Mitchell (references 80 and 81) and by Trumpower and co-workers, is invoked in the following manner (1) One electron is transferred from ubiquinol (ubiquinol oxidized to ubisemi-quinone see Figure 7.27) to the Rieske [2Fe-2S] center at the Qo site, the site nearest the intermembrane space or p side (2) this electron can leave the bci complex via an attached cytochrome c or be transferred to cytochrome Ci (3) the reactive ubisemiquinone reduces the low-potential heme bL located closer to the membrane s intermembrane (p) side (4) reduced heme bL quickly transfers an electron to high-potential heme bn near the membrane s matrix side and (5) ubiquinone or ubisemiquinone oxidizes the reduced bn at the Qi site nearest the matrix or n side. Proton translocation results from the deprotonation of ubiquinol at the Qo site and protonation of ubisemiquinone at the Qi site. Ubiquinol generated at the Qi site is reoxidized at the Qo site (see Figure 7.27). Additional protons are transported across the membrane from the matrix (see Figure 7.26 illustrating a similar process for cytochrome b(6)f). The overall reaction can be written... 

A third, clearer explanation of the electron transfer, proton translocation cycle is given by Saratse. Each ubiquinol (QH2) molecule can donate two electrons. A hrst QH2 electron is transferred along a high-potential chain to the [2Fe-2S] center of the ISP and then to cytochrome Ci. From the cytochrome Cl site, the electron is delivered to the attached, soluble cytochrome c in the intermembrane space. A second QH2 electron is transferred to the Qi site via the cytochrome b hemes, bL and bn. This is an electrogenic step driven by the potential difference between the two b hemes. This step creates part of the proton-motive force. After two QH2 molecules are oxidized at the Qo site, two electrons have been transferred to the Qi site (where one ubiquinone (Qio) can now be reduced, requiring two protons to be translocated from the matrix space). The net effect is a translocation of two protons for each electron transferred to cytochrome c. Each explanation of the cytochrome bci Q cycle has its merits and its proponents. The reader should consult the literature for updates in this ongoing research area. 

Proton gradients can be built up in various ways. A very unusual type is represented by bacteriorhodopsin (1), a light-driven proton pump that various bacteria use to produce energy. As with rhodopsin in the eye, the light-sensitive component used here is covalently bound retinal (see p. 358). In photosynthesis (see p. 130), reduced plastoquinone (QH2) transports protons, as well as electrons, through the membrane (Q cycle, 2). The formation of the proton gradient by the respiratory chain is also coupled to redox processes (see p. 140). In complex III, a Q,cycle is responsible for proton translocation (not shown). In cytochrome c oxidase (complex IV, 3), trans-... 

In chemical terms the photoinduced electron transfer results in transfer of an electron across the photosynthetic membrane in a complex sequence that involves several donor-acceptor molecules. Finally, a quinone acceptor is reduced to a semiquinone and subsequently to a hydroquinone. This process is accompanied by the uptake of two protons from the cytoplasma. The hydroquinone then migrates to a cytochrome be complex, a proton pump, where the hydroquinone is reoxidized and a proton gradient is established via transmembrane proton translocation. Finally, an ATP synthase utilizes the proton gradient to generate chemical energy. Due to the function of tetrapyrrole-based pigments as electron donors and quinones as electron acceptors, most biomimetic systems utilize some... 

The Q cycle accommodates the switch between the two-electron carrier ubiquinone and the one-electron carriers—cytochromes b562, b566, clt and c—and explains the measured stoichiometry of four protons translocated per pair of electrons passing through the Complex III to cytochrome c. Although the path of electrons through this segment of the respiratory chain is complicated, the net effect of the transfer is simple QH2 is oxidized to Q and two molecules of cytochrome c are reduced. 

This basic idea accounts well for the proton translocation that occurs in complex III. In the Q cycle (see fig. 14.11), UQH2 is oxidized to UQ on the outside of the inner membrane, and UQ is reduced to UQH2 on the matrix side. Hydrogen atoms move across the membrane by the diffusion of UQH2 from one of these catalytic sites to the other. The net transfer of two electrons from UQH2 to cytochrome c results in the uptake of two protons from the matrix and release of four protons to the intermembrane space. 

In purple photosynthetic bacteria, electrons return to P870+ from the quinones QA and QB via a cyclic pathway. When QB is reduced with two electrons, it picks up protons from the cytosol and diffuses to the cytochrome bct complex. Here it transfers one electron to an iron-sulfur protein and the other to a 6-type cytochrome and releases protons to the extracellular medium. The electron-transfer steps catalyzed by the cytochrome 6c, complex probably include a Q cycle similar to that catalyzed by complex III of the mitochondrial respiratory chain (see fig. 14.11). The c-type cytochrome that is reduced by the iron-sulfur protein in the cytochrome be, complex diffuses to the reaction center, where it either reduces P870+ directly or provides an electron to a bound cytochrome that reacts with P870+. In the Q cycle, four protons probably are pumped out of the cell for every two electrons that return to P870. This proton translocation creates an electrochemical potential gradient across the membrane. Protons move back into the cell through an ATP-synthase, driving the formation of ATP. 

Transport of two electrons from photosystem II through the cytochrome bhf complex to photosystem I results in the movement of four protons from the chloroplast stroma to the thylakoid lumen. The proton translocation probably occurs in a Q cycle resembling that illustrated in figure 14.11. Two more protons are released in the lumen for each molecule of H20 that is oxidized to 02, and one additional proton is removed from the stroma for each molecule of... 

Electron flow through the cytochrome b6f complex results in proton translocation from the stroma to the thylakoid lumen. In addition, protons are released in the lumen when H20 is oxidized and are taken up from the stromal space when NADP+ is reduced. Protons move from the thylakoid lumen back to the stroma through an ATP-synthase, driving the formation of ATP. 

Krab, K. Wikstrom, M. (1987). Principles of coupling between electron transfer and proton translocation with special reference to proton-translocation mechanisms in cytochrome oxidase. Biochim. Biophys. Acta 895,25-39. 

Fig. 5.3. The major components involved in mitochondrial NADH oxidation in facultative anaerobic mitochondria. In anaerobically functioning mitochondria, NADH is oxidized either by soluble enzymes (left) or by membrane-bound complexes of the electron-transport chain (middle). Under aerobic conditions, a classic respiratory chain is used to oxidize NADH (right). Proton translocation is indicated by H with arrows. Ovals represent the electron transporters RQ, UQ and cytochrome c (cyt. c), and electron transport is indicated by dashed arrows. The vertical bar represents a scale for the standard redox potentials in millivolts. Fum fumarate, NADH-DH NADH dehydrogenase, NADH-ECR soluble NADH enoyl-CoA reductase, RQH2 rhodoquinol, Succ succinate, UQH2 ubiquinol...

A close relationship exists (correlation coefficient = 0.98) between light-induced proton translocation coupled to noncyclic electron flow, with either NADP1 or ferricyanide as Hill reagent, and the percentage of cytochrome b-559 in its HP form. 

Complex III catalyzes electron transfer from reduced coenzyme Q to cytochrome c this process is coupled to vectorial proton translocation with an H+/e stoichiometry of 2. 

Complex IV catalyzes electron transfer from cytochrome c to O2 this process appears to be coupled to proton translocation, with an H+/e value of 2. Two models have been developed to account for these values (Fig. 14-7). Current understanding is that complex IV is capable of acting as a proton pump. 

Backgren, C., Hummer, G., Wiksstroem, M, and Puustinen, A. (2000) Proton translocation by cytochrome c oxidase can take place without conserved glutamic acid in subunit I., Biochemistry, 39, 7863-7867. 

In the binuclear haem-copper centre of cytochrome oxidases there is no cation radical formed at the active site. Instead the extra positive charge is held by the copper atom as it converts from cuprous (Cu1+) to cupric (Cu2+). In fact there is growing evidence to support the model of Mitchell [56] that it is the protonation steps associated with oxidation state changes in this copper atom (Cub) that provide the link between the electron transfer and proton translocation activities of this enzyme. 

Cytochrome oxidases. Mitochondrial cytochrome c oxidase uses the energy involved in the oxidation of cytochrome c and reduction of water to generate a proton electrochemical gradient across the inner mitochondrial membrane [57], As stated above, a ferryl state is an essential intermediate in this process. Similar intermediates are to be expected in all similar proton-translocating cytochrome oxidases that contain a binuclear haem-copper centre,... 

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