Rate Of Proton Uptake Research Articles

Comparison of proton transfer paths to the QA and QB sites of the Rb. sphaeroides photosynthetic reaction centers.

The photosynthetic bacterial reaction centers from purple non-sulfur bacteria use light energy to drive the transfer of electrons from cytochrome c to ubiquinone. Ubiquinone bound in the QA site cycles between quinone, QA, and anionic semiquinone, QA·-, being reduced once and never binding protons. In the QB site, ubiquinone is reduced twice by QA·-, binds two protons and is released into the membrane as the quinol, QH2. The network of hydrogen bonds formed in a molecular dynamics trajectory was drawn to investigate proton transfer pathways from the cytoplasm to each quinone binding site. QA is isolated with no path for protons to enter from the surface. In contrast, there is a complex and tangled network requiring residues and waters that can bring protons to QB. There are three entries from clusters of surface residues centered around HisH126, GluH224, and HisH68. The network is in good agreement with earlier studies, Mutation of key nodes in the network, such as SerL223, were previously shown to slow proton delivery. Mutational studies had also shown that double mutations of residues such as AspM17 and AspL210 along multiple paths in the network presented here slow the reaction, while single mutations do not. Likewise, mutation of both HisH126 and HisH128, which are at the entry to two paths reduce the rate of proton uptake.

Microalgal photosynthesis induces alkalization of aquatic environment as a result of H+ uptake independently from CO2 concentration – New perspectives for environmental applications

The photosynthetic process in microalgae and the extracellular proton environment interact with each other. The photosynthetic process in microalgae induces a pH increase in the aquatic environment as a result of cellular protons uptake rather than as an effect of CO2 consumption. The photosynthetic water photolysis and the reduction/oxidation cycle of the plastoquinone pool provide lumen with protons. Weak bases act as “permeant buffers” in lumen during the photosynthetic procedure, converting the ΔpH to Δψ. This is possibly the main reason for continuous light-driven proton uptake from the aquatic environment through cytosol and stroma, into the lumen. The proton uptake rate and, therefore, the microalgal growth is proportional to the light intensity, cell concentration, and extracellular proton concentration. The low pH in microalgae cultures, without limitation factors related to light and nutrients, strongly induces photosynthesis (and proton uptake) and, consequently, growth. In contrast, the mitochondrial respiratory process, in the absence of photosynthetic activity, does not substantially alter the culture pH. Only after intensification of the respiratory process, using exogenous glucose supply leads to significantly reduced pH values in the culture medium, almost exclusively through proton output. Enhanced dissolution of atmospheric CO2 in water causes the phenomenon of ocean acidification, which prevents the process of calcification, a significant process for numerous phytoplankton and zooplankton organisms, as well for corals. The proposed interaction between microalgal photosynthetic activity and proton concentration in the aquatic environment, independently from the CO2 concentration, paves the way for new innovative management strategies for reversing the ocean acidification.

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

Role of subunit III and its lipids in the molecular mechanism of cytochrome c oxidase

The terminal respiratory enzyme cytochrome c oxidase (CcO) reduces molecular oxygen to water, and pumps protons across the inner mitochondrial membrane, or the plasma membrane of bacteria. A two-subunit CcO harbors all the elements necessary for oxygen reduction and proton pumping. However, it rapidly undergoes turnover-induced irreversible damage, which is effectively prevented by the presence of subunit III and its tightly bound lipids. We have performed classical atomistic molecular dynamics (MD) simulations on a three-subunit CcO, which show the formation of water wires between the polar head groups of lipid molecules bound to subunit III and the proton uptake site Asp91 (Bos taurus enzyme numbering). Continuum electrostatic calculations suggest that these lipids directly influence the proton affinity of Asp91 by 1–2pK units. We surmise that lipids bound to subunit III influence the rate of proton uptake through the D-pathway, and therefore play a key role in preventing turnover-induced inactivation. Atomistic MD simulations show that subunit III is rapidly hydrated in the absence of internally bound lipids, which is likely to affect the rate of O2 diffusion into the active-site. The role of subunit III with its indigenous lipids in the molecular mechanism of CcO is discussed.

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ∼5,000. Here, we show that proton uptake at full speed (∼10(4) s(-1)) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn(2+) addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn(2+) binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

Proton Uptake and pKaChanges in the Uncoupled Asn139Cys Variant of CytochromecOxidase

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O(2) to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located ~10 Å from the protein surface, is particularly sensitive to mutations that uncouple the O(2) reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

The Membrane Modulates Internal Proton Transfer in Cytochrome c Oxidase

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O(2) reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate. This pK(a) change resulted in decreased proton-uptake rates in the pH range of ~6.5-9.5, which is explained in terms of lowering of the pK(a) of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal Cu(A)-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.

Amphiphilic block copolymers significantly influence functions of bacteriorhodopsin in water

This paper investigates the effects of a macromolecular amphiphile poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO–PPO–PEO) on a photoresponsive membrane protein, bacteriorhodopsin (BR). After incubation of BR in EO23–PO65–EO23 (P123) solution, BR maintained its function as a light-driven proton pump; however, the rate of proton uptake and lifetime of the M intermediate in the photocycle of BR upon illumination were, under appropriate conditions, prolonged by about three orders of magnitude compared with that of native BR, even at neutral pH. Measurements using circular dichroism spectroscopy and dynamic light scattering indicated that BR molecules were still in a trimer state after treatment with the copolymers. This is quite different to BR, which showed a much slower photoresponse during drying. The BR–P123 assemblies did not exhibit significantly different photoresponsive behavior with changes to the water content, which implied that in the case of dried BR films or dried BR–polymer films, the elongation of the M decay may be caused not by lack of water molecules necessary for proton transfer, but by protein immobilization. Determination of the critical micelle concentration of P123 with and without BR revealed that this prolongation effect is closely related to the formation of micelles. The above phenomenon was also observed with 6 other Pluronic copolymers. In solutions of small molecular detergents, such as Triton X-100, the photoresponse of BR was prolonged as well; the extent of prolongation was, however, much less than in solutions of macromolecular amphiphiles. The formation of a local polymer coating due to self assembly of the copolymer and protein molecules might be responsible for this very significant prolongation effect, which is beneficial for the potential application of BR as an information material.

Surface Proton Donors for the D-Pathway of CytochromecOxidase in the Absence of Subunit III

The major proton-transfer pathway into the buried active site of cytochrome c oxidase (CcO) is the D-pathway that begins with the subunit I residue Asp-132 on the inner protein surface (the cytoplasmic surface of the aa3-type CcO of Rhodobacter sphaeroides). Asp-132 is surrounded by residues from both subunits I and III. In the absence of subunit III, CcO retains activity, but the functional characteristics of the D-pathway are significantly altered such that the transfer of protons from Asp-132 into the pathway becomes the rate-limiting step. Determination of the pH-dependence of the rate constant for D-pathway proton uptake during the single-turnover of CcO indicates that the pKa of Asp-132 in the absence of subunit III is approximately 7. The removal of subunit III also allows for alternative surface proton donor/acceptors other than Asp-132. With Asp-132 altered to alanine, the rate constant for D-pathway proton uptake is very slow (5 s(-1)) in the presence of subunit III. Once subunit III is removed, the proton uptake rate constant increases 80-fold, to 400 s(-1). The pKa associated with this uptake is >10, and the initial proton donor/acceptor in D132A III (-) is proposed to be a water of the D-pathway rather than an amino acid residue. Arachidonic acid (Aa), which stimulates the activity of several D-pathway mutant CcOs, appears to become the initial proton donor/acceptor in the absence of subunit III, whether or not Asp-132 is altered. Aa shifts the pKa of the initial proton donor to 7.6 for both wild-type (WT) III (-) and D132A III (-). The results indicate that subunit III creates a barrier that helps prevent protons from donors other than Asp-132 from directly accessing the internal waters of the D-pathway, while the subunit also provides an environment that increases the rate at which Asp-132 transfers protons into the D-pathway.

Determination of proton transfer rates by chemical rescue: application to bacterial reaction centers.

The bacterial reaction center (RC) converts light into chemical energy through the reduction of an internal quinone molecule Q(B) to Q(B)H(2). In the native RC, proton transfer is coupled to electron transfer and is not rate-controlling. Consequently, proton transfer is not directly observable, and its rate was unknown. In this work, we present a method for making proton transfer rate-controlling, which enabled us to determine its rate. The imidazole groups of the His-H126 and His-H128 proton donors, located at the entrance of the transfer pathways, were removed by site-directed mutagenesis (His --> Ala). This resulted in a reduction in the observed proton-coupled electron transfer rate [(Q(A)(-)(*)Q(B))Glu(-) + H(+) --> (Q(A)Q(B)(-)(*))GluH], which became rate-controlled by proton uptake to Glu-L212 [Adelroth, P., et al. (2001) Biochemistry 40, 14538-14546]. The proton uptake rate was enhanced (rescued) in a controlled fashion by the addition of imidazole or other amine-containing acids. From the dependence of the observed rate on acid concentration, an apparent second-order rate constant k((2)) for the "rescue" of the rate was determined. k((2)) is a function of the proton transfer rate and the binding of the acid. The dependence of k((2)) on the acid pK(a) (i.e., the proton driving force) was measured over 9 pK(a) units, resulting in a Brönsted plot that was characteristic of general acid catalysis. The results were fitted to a model that includes the binding (facilitated by electrostatic attraction) of the cationic acid to the RC surface, proton transfer to an intermediate proton acceptor group, and subsequent proton transfer to Glu-L212. A proton transfer rate constant of approximately 10(5) s(-)(1) was determined for transfer from the bound imidazole group to Glu-L212 (over a distance of approximately 20 A). The same method was used to determine a proton transfer rate constant of 2 x 10(4) s(-)(1) for transfer to Q(B)(-)(*). The relatively fast proton transfer rates are explained by the presence of an intermediate acceptor group that breaks the process into sequential proton transfer steps over shorter distances. This study illustrates an approach that could be generally applied to obtain information about the individual rates and energies for proton transfer processes, as well as the pK(a)s of transfer components, in a variety of proton translocating systems.

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 μM, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO2-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 μM CO2) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO2-dependent O2evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO2-dependent O2evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C3-plants are discussed.

Identification of the proton pathway in bacterial reaction centers: both protons associated with reduction of QB to QBH2 share a common entry point.

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, Q(B). This process involves the transfer of two protons from the aqueous solution to the protein-bound Q(B) molecule. The second proton, H(+)(2), is supplied to Q(B) by Glu-L212, an internal residue protonated in response to formation of Q(A)(-) and Q(B)(-). In this work, the pathway for H(+)(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of Q(A) (DQ(A)-->D(+)Q(A)(-)) and Q(B) (DQ(B)-->D(+)Q(B)(-)), studied by using pH-sensitive dyes. The other was the electron transfer k(AB)((1)) (Q(A)(-)Q(B)-->Q(A)Q(B)(-)). At pH 8.5, binding of Zn(2+), Cd(2+), or Ni(2+) reduced the rates of proton uptake upon Q(A)(-) and Q(B)(-) formation as well as k(AB)((1)) by approximately an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D(+)Q(A)(-) is formed 10(5)-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212-->Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn(2+) binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H(+)(2) was thus identified to be the same as for the first proton H(+)(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Proton stoichiometry of the overexpressed uracil symport of the yeast Saccharomyces cerevisiae.

Information about the molecular biology of the uracil carrier of Saccharomyces cerevisiae is now available but its properties as a symport are unexplored. We have now studied its proton stoichiometry at pH 6.5, 4.8 and 4.2, in a yeast strain overexpressing the symport and unable to metabolize uracil. After the depletion of cellular ATP, uracil uptake followed an approximately exponential time course to reach a plateau. Proton uptake was then indistinguishable from the basal rate shown in controls without added uracil. It was concluded that more than 87% of the uracil flux through the system was coupled to symported protons. Because the basal rate was a significant fraction of the total rate of proton uptake at pH 4.2 or 4.8, the ratio of the proton and uracil flows could not be defined uniquely during the initial near-linear phase of uptake. However, the average rate of proton uptake during increasing time intervals up to 120 s was shown to be a linear function of the corresponding average rate of uracil uptake. This relationship, defining approx. 90% of the eventual uracil uptake, was used to deduce the mean proton stoichiometry as approx. 3 at pH 4.2, falling to near 2 at pH 6.5. The rate of uracil uptake increased 4-fold when the negative proton gradient (Delta mu(H)) acting across the plasma membrane increased from 60 to 200 mV. The rate fell markedly after depolarization by KCl. The maximum uracil gradient (Delta mu(S)) was less affected by depolarization. The ratio Delta mu(S)/Delta mu(H) fell from approx. 2 to near 1 as Delta mu(H) increased in the above range. In contrast with the starved yeast, uracil accumulation during energy metabolism followed a pump-leak model.

Inhibition of proton transport through spinach thylakoid membranes by 5,5'-dithiobis(2-nitrobenzoate).

In both oxidative phosphorylation and photo-phosphorylation, electron flow through a carrier is linked to the generation of ATP. The energy released by electron transport is converted to potential energy forming a proton gradient across the membranes in chloroplasts. The proton gradient can be measured by a pH microelectrode. In this report, pH changes produced by photo-induced proton transport through spinach chloroplast membranes were measured by a glass microelectrode. The effect of 5,5'-dithiobis(2-nitrobenzoate) (DTNB) on the kinetics of proton movement across the thylakoid membranes was studied. The results showed that the rate of proton uptake was reduced with increasing DTNB concentration. The rate of leakage of accumulated protons through thylakoid membranes also decreased. The results support the notion that cysteinyl residue is involved in proton translocation. The inhibition of proton transport would subsequently affect the chemical reactions of the Calvin Cycle that takes place in the stroma which is the soluble compartment surrounding the thylakoid membranes.

Effect of pyrrolidine dithiocarbamate on photo-induced proton transport through chloroplast membranes.

pH changes produced by photo-induced proton transport through chloroplast membranes in spinach were measured by a glass microelectrode. Effect of pyrrolidine dithiocarbamate on proton translocation through chloroplast membranes has been studied. Kinetic analysis of proton translocation shows that the rate is reduced as the carbamate concentration increases. The rate of proton uptake follows first-order kinetics and diminishes with increasing carbamate concentrations. The outward leakage of accumulated protons through thylakoid membranes in the dark also decreases likewise. However, the leakage of protons takes a much longer time. Pyrrolidine dithiocarbamate is an effective inhibitor of proton transport through chloroplast membranes. The results suggest that the photo-induced proton translocation is regulated by conformation change in the membrane. Higher concentration of carbamate disrupts the tertiary conformation of the membrane. The inhibition of proton transport would affect ATPase function; thus, an excess use or accumulation of pyrrolidine thiocarbamate may compromise ATP production.

Interaction of light metals and protons with seaweed biosorbent

Based on their displacement by protons, the following ascending order of light metals affinity toward Sargassum fluitans biomass was observed: Na + ⩽ K + < Mg 2+ < Ca 2+ < Al 3+. Higher biomass-metal affinities resulted in lowering the leaching tendency of (polysaccharidic) biomass components during the sorption process. More than 12% weight loss of protonated biomass occurred during 2 min of sorption, which should be considered in all stoichiometric and sorption calculations. The rate of proton uptake for Na-loaded and Ca-loaded S. fluitans biomass at pH 4.5 was lower than the rate of desorption at pH 1.1. In the case of Na-loaded S. fluitans, the predominant proton uptake rate was of a zero order. Na uptake by protonated S. fluitans biomass did not take place at all in the presence of Ca and Al ions. However, it increased in the absence of other light metals. Since the affinity of Ca 2+ for the biomass was much greater than that of Na +, the three-component system of Na +, H + and Ca 2+ could be simplified into proton-calcium ion exchange. The initial rate of Ca uptake with protonated S. fluitans biomass at pH 4.5 was lower than that of proton release from biomass. Approximately 20% of Ca 2+ sorbed was bound to the binding site as a mono-valent ion. Titration and stoichiometric indications pointed to the fact that Al was being sorbed as a hydroxyl complex.

Paraquat Affects Light-Induced Proton Transport Through Chloroplast Membranes in Spinach

In both oxidative phosphorylation and photo-phosphorylation, electron flow through a carrier is linked to the generation of ATP. The energy released by electron transport is converted to potential energy forming a proton gradient across the membranes in chloroplasts and that is coupled to ADP phosphorylation. In this report, a method is used to study effects of paraquat on proton movement across the chloroplast membranes. Paraquat is a bipyridyl herbicide and its salt is used as a biological oxidation-reduction indicator. Light-induced proton transport through chloroplast thylakoids is affected by the presence of paraquat. The proton uptake upon illumination and proton leakage in the dark were enhanced at a low paraquat concentration (< 20 nM). The rate of proton uptake by the illuminated thylakoids followed the first order of kinetics. However, paraquat gradually reduces proton transport with the increase of the herbicide concentration. The pH at the steady state in the illuminated thylakoid membranes becomes more acidic with the elevation of the herbicide concentration. The method is useful in the study of the toxic effect of herbicides on the fundamental process of photosynthesis.

Oligomeric Regulation of Gastric H+,K+-ATPase

The H+,K+-ATPase of intact gastric vesicles has two Km values for ATP hydrolysis, 7 and 80 microM. Irradiation of vesicles with ultraviolet light in the presence of 1 mM ATP resulted in K+-ATPase activity that shows only the low affinity ATP binding. The irradiation stimulated or inhibited proton uptake rate compared with control vesicles at high or low ATP concentrations, respectively. The relation between proton uptake rate and K+-ATPase activity at different ATP concentrations was linear with irradiated vesicles and nonlinear with control vesicles. These results indicate that hydrolysis at the high affinity ATP binding site regulates the energy-transport coupling in negative and positive manners at high and low ATP concentrations, respectively. The complete inhibition of K+-ATPase by a specific proton pump inhibitor E3810 (rabeprazole) (2-([4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulf i nyl)-1H-benzimidazole sodium salt) occurred when E3810 bound to half of the alpha-subunit of H+,K+-ATPase in unirradiated vesicles at both 200 and 10 microM ATP, whereas the complete inhibition of proton uptake occurred when E3810 bound to half or a quarter of the alpha-subunit at 200 or 10 microM ATP, respectively. These results suggest that dimeric interaction between the alpha-subunits is necessary for the enzyme activity at all ATP concentrations and that dimeric or tetrameric interaction is necessary for proton transport at high or low ATP concentrations, respectively.

Control of electron delivery to the oxygen reduction site of cytochrome c oxidase: A role for protons

We have studied the reaction of oxidized "pulsed" cytochrome c oxidase with reduced cytochrome c and with ruthenium(II) hexaammine using stopped-flow mixing. The rate of reduction of Fea3 (the oxygen-binding heme) is not a linear function of the population of reduced Fea (the low-spin heme), as would be expected if electron transfer between these sites is rate-limiting. Instead, the rate can be increased significantly by increasing the driving force of the reductant (lowering of Eh) even after Fea is almost completely reduced. Reduction of Fea3 becomes slower as the pH is raised, and consumption of protons can be seen simultaneously with electron entry into Fea3. Both the reduction of Fea3 and the proton uptake are biphasic. To explain these findings, we propose a model in which (1) intramolecular heme-heme electron transfer is fast, and has an essentially constant rate; (2) when reduction begins, the midpoint potentials of Fea3 and CuB are initially low, and only a small fraction of these centers become reduced; and (3) this reduced population is then stabilized by the uptake of protons. Thus, net reduction of Fea3 and CuB is controlled by the amount of the low-potential population which becomes reduced together with the rate of proton uptake by this reduced low-potential species. Important consequences of this mechanism for the function of the enzyme and for the respiratory chain as a whole are discussed.

Oxygen Reaction and Proton Uptake in Helix VIII Mutants of Cytochrome bo3

The oxygen reaction of wild-type and helix VIII mutants of cytochrome bo3 from Escherichia coli, and the associated proton uptake during this reaction, has been studied using flash photolysis of the CO complex of the reduced protein after rapid mixing with oxygen. We have focused on mutations in the transmembrane helix VIII where protonatable residues have been exchanged, and mainly on the inactive mutants (i.e., T352A, T359A, and K362L, -M, and -Q). The kinetics for electron transfer during oxidation for the mutants are similar to the wild-type; two rate constants of 3.2 x 10(4) and 3.4 x 10(3) s-1 (at 1 mM oxygen) are detected. Proton uptake is observed for wild-type as well as for the mutant enzymes, but the mutations within helix VIII have affected the rate of proton uptake; it is significantly accelerated in the mutants. These results show that none of the protonatable residues in helix VIII are required in the reaction between the fully reduced cytochrome bo3 and oxygen. We have also studied electron redistribution after photolysis of CO from the mixed-valence compound; we found three kinetic components for wild-type and the mutants T352A and T359A, but for K362M only the first and third components are observed, with amplitudes that are lower than those for the corresponding components in the wild-type enzyme, suggesting that the characteristics of internal electron transfer in the K362M mutant are different from those of the wild-type enzyme.