Potential proton translocators

The third alternative is proton exchange along hydrogen-bonded water molecules (33-35). In bacteriorhodopsin, for example, a recent structural model at 3.5-A resolution strongly suggests that water molecules form a narrow channel and are involved in proton delivery to the chromophore (36). The remainder of this review will discuss chains of hydrogen-bonded water molecules as potential proton translocators and describe some initial tests of the concept. 

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).

The electrochemical potential difFetence across the membrane, once established as a tesult of proton translocation, inhibits further transport of teducing equivalents through the respiratory chain unless discharged by back-translocation of protons across the membtane through the vectorial ATP synthase. This in turn depends on availability of ADP and Pj. 

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. 

The xanthenes exist in solution in several different forms depending on pH, as shown in Figure 2 and Table 1 [18]. The emission quantum yield of fluorescein depends on the acidity of the solution, the fluorescence intensity decreasing as the protonated forms of the dye come to predominate with decreasing pH. This pH sensitivity allows fluorescein derivatives to be employed as pH indicators, to measure the pH inside living cells [19-22], at water-lipid interfaces [23], and in the interior of phospholipid vesicles [24]. The sensitivity of fluorescein emission to the pH of the medium has also been used to measure lateral proton conductances at water-lipid interfaces [25-28] and proton translocation across phospholipid vesicles [29] and to determine the electrostatic potential of macromolecules [30, 31]. The pheno-... 

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. 

Extensive studies have been carried out on the proton-translocating ATPase of mitochondrial, bacterial and chloroplast membranes. This enzyme can also function in reverse to exploit the electrochemical potential of protons built up by respiration for the synthesis of ATP from ADP and P .298 The synthesis of ATP can be effected by the application of external electrical pulses to the ATPase vesicles in suspension with submitochondrial particles, showing that the diffusion potential of the protons (ApH) is not used. The yield of ATP was linearly dependent on the number of pulses.299... 

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...

The chemiosmotic model requires that flow of electrons through the electron-transport chain leads to extrusion of protons from the mitochondrion, thus generating the proton electrochemical-potential gradient. Measurements of the number of H+ ions extruded per O atom reduced by complex IV of the electron-transport chain (the H+/0 ratio) are experimentally important because the ratio can be used to test the validity of mechanistic models of proton translocation (Sec. 14.6). 

Tight coupling between ATP synthesis and proton translocation is dependent on the impermeability of the membrane to protons, so that the F0 channel and ATP synthesis provide the only way for protons to reenter the mitochondrial matrix. Physical damage to the membranes, or chemicals that allow the dissipation of the proton or electrical potential gradient, will allow alternative pathways for reentry of protons, and will uncouple respiration from ATP synthesis (see Problem 14.5). 

The isomerization models discussed above differ from that described by Warshel, where J625 and PBAT are partially isomer-ized chromophores (90°) and their decay to Kfc g and BAT, respectively, is described by a motion on a potential surface involving both protein relaxation (proton translocation) and additional chromophore isomerization. This model implies that in K ig and BAT, proton translocation has taken place in the opsin, but it is not discriminative concerning whether a chromophore isomerization has taken place at this stage. 

The reaction centre found in many purple non-sulphur bacteria is a simple example of a group of proteins that are natureis solar batteries. The reaction centre uses the energy of sunlight to generate positive and negative charges on opposite sides of the bacterial cytoplasmic membrane. This potential difference drives a circuit of electron transfer reactions that are linked to proton translocation across this membrane. 

Spectroscopic and crystallographic studies have identified four Fe S clusters in the membrane-bound photosynthetic electron transport chain of plant and cyanobacterial chloro-plasts. One is the Rieske-type [2Fe-2S] + + center in the cyt b(,f complex, which catalyzes electron transfer from plasto-quinol to plastocyanin with concomitant proton translocation, and is functionally analogous to the cyt bc complex, with cyt / in place of cyt The remainder are low-potential [4Fe 4S] + + centers in Photosystem I which constitute the terminal part of the electron transfer chain that is initiated by the primary donor chlorophyll. One is a very low-potential [4Fe S] + + center, Fx (Em =-705 mV), that bridges two similar subunits (PsaA and PsaB) and is coordinated by two cysteines from each subunit in a C-Xg-C arrangement. This cluster transfers electrons to the 2Fe-Fd acceptor via an electron transfer chain composed of Fa, a [4Fe S] + + cluster with Em = -530 mV, and Fb, a [4Fe S] + + clusters with Em = -580 mV. Fa and Fb are in a low-molecular weight subunit (PsaC, 9 kDa) that shows strong sequence and structural homology with bacterial 8Fe-Fds. The center-to-center distance between Fx and Fa and between Fa and Fb are 14.9 A and 12.3 A, respectively, well... 

In this way, the loss of redox free energy occurring during electron transport is partially conserved as electrochemical potential energy of the proton gradient. The synthesis of ATP occurs when the protons accumulated inside the thylakoid lumen are transported out into the external water phase by an anisotropic, proton-translocating ATP synthase-ATPase (the complex CFq-CFi), which catalyses the reaction... 

An electron moves from to Qg in about 200 p,s [28-31,51]. Excitation of the reaction center by a second photon sends another electron from P to Q, and then on to Qg with similar kinetics. The fully reduced Qg now probably picks up two protons from the solvent, dissociates from the reaction center as the quinol (QgH2), and is replaced by a fresh molecule of ubiquinone. Electrons from OgH2 return to P" via a Cyt bc complex and a high-potential, c-type cytochrome. This cyclic electron flow drives proton translocation across the chromatophore membrane, and is coupled to the formation of ATP. 

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