Direct measurement of electric current generation by cytochrome oxidase, H+-ATPase and bacteriorhodopsin.

Drachev, Lel A.; Jasaitis, Audrius; Kaulen, Andrey D.; Kondrashin, Andrey A.; Liberman, E. A.; Nemecek, I. B.; Остроумов, С. А.; Semenov, Alexey Yu.; Skulachev, V. P.

doi:10.1038/249321a0

Cited by 237 publications

(83 citation statements)

References 13 publications

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“…Previously, the assay was used to demonstrate generation of dly by COX in proteoliposomes [20] or submitochondrial particles [21] under steady-state conditions. Here we describe the association of COV with phospholipid-impregnated collodion film and report on the rapid electrogenic processes linked to flash-induced reduction of COX Compound F to the Ox form by RuBpy.…”

Section: Chemicals Preparationsmentioning

confidence: 99%

Flash‐induced membrane potential generation by cytochrome c oxidase

et al. 1993

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Flash‐induced single‐electron reduction of cytochrome c oxidase. Compound F (oxoferryl state) by RuII(2,2'‐bipyridyl)2+ 3 [Nilsson (1992) Proc. Natl. Acad. Sci. USA 89, 6497‐6501] gives rise to three phases of membrane potential generation in proteoliposomes with τ values and contributions of ca. 45 μs (20%), 1 ms (20%) and 5 ms (60%). The rapid phase is not sensitive to the binuclear centre ligands, such as cyanide or peroxide, and is assigned to vectorial electron transfer from CuA to heme a. The two slow phases kinetically match reoxidation of heme a, require added H2O2 or methyl peroxide for full development, and are completely inhibited by cyanide; evidently, they are associated with the reduction of Compound F to the Ox state by heme a. The charge transfer steps associated with the F to Ox conversion are likely to comprise (i) electrogenic uptake of a ‘chemical’ proton from the N phase required for protonation of the reduced oxygen atom and (ii) electrogenic H+ pumping across the membrane linked to the F to Ox transition. Assuming heme a ‘electrical location’ in the middle of the dielectric barrier, the ratio of the rapid to slow electrogenic phase amplitudes indicates that the F to Ox transition is linked to transmembrane translocation of 1.5 charges (protons) in addition to an electrogenic uptake of one ‘chemical’ proton required to form Fe3+‐OH− from Fe4+ = O2−. The shortfall in the number of pumped protons and the biphasic kinetics of the millisecond part of the electric response matching biphasic reoxidation of heme a may indicate the presence of 2 forms of Compound F, reduction of only one of which being linked to full proton pumping.

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Section: Chemicals Preparationsmentioning

confidence: 99%

Flash‐induced membrane potential generation by cytochrome c oxidase

et al. 1993

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“…In this respect, it is tempting to consider data from electrometric measurements that have been used to study time-dependent membrane potentials and to elucidate charge motions in membrane proteins. Such approaches can offer mechanistic insights into the function of membrane-bound channels and pumps (32). Monitoring the transmembrane potential may enable observation of PT steps that are typically "invisible" when using spectroscopic techniques.…”

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confidence: 99%

Analyzing the electrogenicity of cytochrome c oxidase

Kim

Warshel

2016

Proc. Natl. Acad. Sci. U.S.A.

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Measurements of voltage changes in response to charge separation within membrane proteins can offer fundamental information on spectroscopically "invisible" steps. For example, results from studies of voltage changes associated with electron and proton transfer in cytochrome c oxidase could, in principle, be used to discriminate between different theoretical models describing the molecular mechanism of proton pumping. Earlier analyses of data from these measurements have been based on macroscopic considerations that may not allow for exploring the actual molecular mechanisms. Here, we have used a coarse-grained model describing the relation between observed voltage changes and specific charge-transfer reactions, which includes an explicit description of the membrane, the electrolytes, and the electrodes. The results from these calculations offer mechanistic insights at the molecular level. Our main conclusion is that previously assumed mechanistic evidence that was based on electrogenic measurements is not unique. However, the ability of our calculations to obtain reliable voltage changes means that we have a tool that can be used to describe a wide range of electrogenic charge transfers in channels and transporters, by combining voltage measurements with other experiments and simulations to analyze new mechanistic proposals.electrogenicity | membrane potential | proton transfer | electron transfer C ytochrome c oxidase (CcO) couples the four-electron reduction of O 2 to water, where the released free energy is used to pump protons from the negative (N) to the positive (P) side of the membrane (1-5), leading to an electrochemical proton gradient that drives, for example, ATP synthesis. The elucidation of the structure of CcO (6-12), combined with experimental and theoretical studies (e.g., refs. 3, 13-15), has advanced the understanding of this intriguing system. Progress has been made in defining the conditions that would allow CcO to pump protons against a pH gradient (4, 16), in estimating the electrostatic energy of possible intermediates (17-21), in evaluating the energetics of the key water chains (22,23), and of a number of specific proton-transfer (PT) reactions (16). Furthermore, examination of the energetics of the overall pumping process has been performed by using a semimacroscopic model (16).However, the relationship between protein structure, the PT energetics, and detailed PT trajectories has not been established. Furthermore, to our knowledge, a consistent protonpumping mechanism purely based on structural information and thermodynamic considerations has not been presented. For example, we still cannot identity the primary acceptor for pumped protons, although the D propionate of heme a 3 (Prda3 in Fig. 1) is one of the most likely candidates for being at least a "transient" acceptor. Apparently, there has been some progress in using structures in functional analyses (e.g., refs. 16, 24-26). However, we still do not have a consistent model that correctly reproduces the pumping events, while prev...

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“…2 and 7). The pH gradient is used by the bacteria to drive the synthesis of ATP and is a much simpler photosynthetic system than the electron pumping mechanism in chlorophyll (8,9).…”

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The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

Heyes

El‐Sayed

2002

Journal of Biological Chemistry

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We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65°C, and the premelting transition in which the protein changes conformation in native bR at 80°C could not be observed. Also, the N-H to N-D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91°C compared with 80°C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91°C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.

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Direct measurement of electric current generation by cytochrome oxidase, H+-ATPase and bacteriorhodopsin.

Cited by 237 publications

References 13 publications

Flash‐induced membrane potential generation by cytochrome c oxidase

Flash‐induced membrane potential generation by cytochrome c oxidase

Analyzing the electrogenicity of cytochrome c oxidase

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

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