Kinetics Of Proton Uptake Research Articles

Electron and proton transfer in the M. smegmatis III2IV2 supercomplex

The M. smegmatis respiratory III2IV2 supercomplex consists of a complex III (CIII) dimer flanked on each side by a complex IV (CIV) monomer, electronically connected by a di-heme cyt. cc subunit of CIII. The supercomplex displays a quinol oxidation‑oxygen reduction activity of ~90 e−/s. In the current work we have investigated the kinetics of electron and proton transfer upon reaction of the reduced supercomplex with molecular oxygen. The data show that, as with canonical CIV, oxidation of reduced CIV at pH 7 occurs in three resolved components with time constants ~30 μs, 100 μs and 4 ms, associated with the formation of the so-called peroxy (P), ferryl (F) and oxidized (O) intermediates, respectively. Electron transfer from cyt. cc to the primary electron acceptor of CIV, CuA, displays a time constant of ≤100 μs, while re-reduction of cyt. cc by heme b occurs with a time constant of ~4 ms. In contrast to canonical CIV, neither the P → F nor the F → O reactions are pH dependent, but the P → F reaction displays a H/D kinetic isotope effect of ~3. Proton uptake through the D pathway in CIV displays a single time constant of ~4 ms, i.e. a factor of ~40 slower than with canonical CIV. The slowed proton uptake kinetics and absence of pH dependence are attributed to binding of a loop from the QcrB subunit of CIII at the D proton pathway of CIV. Hence, the data suggest that function of CIV is modulated by way of supramolecular interactions with CIII.

Proton uptake kinetics and electromotive force in BaCo0.4Fe0.4Zr0.1Y0.1O3-δ cathode material with e−/O2−/H+ three mobile carriers for protonic ceramic fuel cells

BaCo0.4Fe0.4Zr0.1Y0.1O3-δ (BCFZY) as a mixed electron, oxygen ion, and proton-conducting cathode materials is of particular interest as it leads to lower operation temperature for protonic ceramic fuel cells (PCFCs). This work investigates the proton uptake kinetics and electromotive force of BCFZY material with the predominant proton uptake reactions as hydrogenation by using electrical conductivity relaxation method. The deviation in proton uptake kinetics affected by oxygen partial pressure and water pressure is analyzed. The electromotive force induced by water partial pressure change is calculated based on the Nernst equation. The research implications of electron/oxygen ion/proton triple conductor materials for application in PCFCs have also been discussed.

Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry

Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O2-reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Å from the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme.

Investigate the proton uptake process of proton/oxygen ion/hole triple conductor BaCo0.4Fe0.4Zr0.1Y0.1O3-δ by electrical conductivity relaxation

The proton surface uptake kinetics of proton, oxygen ion and hole triple conductor BaCo0.4Fe0.4Zr0.1Y0.1O3-δ (BCFZY) is investigated by electrical conductivity relaxation method (ECR). The proton uptake process is controlled by changing the water partial pressure at constant temperature. And the proton concentration in BCFZY bulk could be calculated by the conductivity change value (Δσ: 0.063 Scm−1 at 500 °C) from 0% to 10% pH2O. The proton surface exchange coefficient, kδ is 3.85 × 10−6 cm−1 at 500 °C, derived from fitting the relaxation curve. It is also found the proton uptake reaction rate would be enhanced under higher oxygen partial pressure. And the kδ value could be greatly enhanced by one order of magnitude with doped ceria coated on BCFZY surface, which also proved that the proton uptake kinetics is limited by the surface exchange step. In all, this work provides an effective way to measure the proton surface exchange kinetic for proton conductive cathode.

Effects of Mutations of Lys41 and Asp102 of Bacteriorhodopsin

Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK(a) of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK(a) of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.

Volutin Granules of Eimeria Parasites are Acidic Compartments and Have Physiological and Structural Characteristics Similar to Acidocalcisomes

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.

Preparation of silica-supported nanoporous manganese oxides

Manganese oxide micro-crystals were bound onto silica to prepare a composite material with a particle size large enough to be used in a flow-through fixed bed for metal uptake from hydrometallurgical solutions. Commercial β-MnO 2 was used for screening of the optimal composite preparation conditions like pH, gelation temperature, and the SiO 2/MnO 2 ratio. The target composites were made using synthetic nanoporous manganese oxides OL-1 (birnessite) and OMS-1 (todorokite). The resulting silica-supported materials were characterized using scanning electron microscopy, N 2 adsorption measurements, particle size measurements as well as Raman spectroscopy and IR spectroscopy. Proton uptake kinetics of OL-1/SiO 2 and OMS-1/SiO 2 composites was compared with that of the unsupported analogues. Results show that the MnO x /SiO 2 ratio affects significantly the properties of the composite. The higher is the ratio, the lower are the specific surface area, the pore volume and the pore size. On the other hand, higher amount of the silica binder gives composites of better mechanical properties. Thus, a compromise between the mechanical strength and porosity has to be chosen. OL-1 and OMS-1 can be aggregated successfully with colloidal silica at pH 4 and with MnO 2/SiO 2 weight ratio of 0.7. The best gelation and drying temperature was 110 °C and sufficiently strong composites were obtained by additional heat-treatment at 250 °C for 2 h. Determination of the proton sorption kinetics in nitrate media revealed that aggregation of OMS -1 with colloidal silica does not affect significantly the uptake kinetics. However, in the case of OL-1, the aggregation with silica has a negative effect on the uptake rate. The difference is explained by the structure of the composites.

Monitoring The Activation Of Rhodopsin By The Transient Fluorescence Of Fluorescently Labeled Helix 8

The transient changes of the fluorescence of bovine rhodopsin in ROS membranes selectively labeled with Alexa594 at cysteine 316 in helix 8 were followed in time from 1 μs to 10 s after flash excitation of the photoreceptor. A large light-induced transient fluorescence increase was observed with time constants in the ms- range at pH6. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state MII (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6), the sequence of events is: Schiff base deprotonation, structural change, proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

The Bacteriorhodopsin Carboxyl-Terminus Contributes to Proton Recruitment and Protein Stability

We examined functional and structural roles for the bacteriorhodopsin (bR) carboxyl-terminus. The extramembranous and intracellular carboxyl-terminus was deleted by insertion of premature translation stop codons. Deletion of the carboxyl-terminus had no effect on purple membrane (PM) lattice dimensions, sheet size, or the electrogenic environment of the ground-state chromophore. Removal of the distal half of the carboxyl-terminus had no effect on light-activated proton pumping, however, truncation of the entire carboxyl-terminus accelerated the rates of M-state decay and proton uptake approximately 3.7-fold and severely distorted the kinetics of proton uptake. Differential scanning calorimetry (DSC) and SDS denaturation demonstrated that removal of the carboxyl-terminus decreased protein stability. The DSC melting temperature was lowered by 6 degrees C and the calorimetric enthalpy reduced by 50% following removal of the carboxyl-terminus. Over the time range of milliseconds to hours at least 3 phases were required to describe the SDS denaturation kinetics for each bR construction. The fastest phases were indistinguishable for all bR's, and reflected PM solubilization. At pH 7.4, 20 degrees C, and in 0.3% SDS (w/v) the half-times of bR denaturation were 19.2 min for the wild-type, 12.0 min for the half-truncation and 3.6 min for the full-truncation. Taken together the results of this study suggest that the bR ground state exhibits two "domains" of stability: (1) a core chromophore binding pocket domain that is insensitive to carboxyl-terminal interactions and (2) the surrounding helical bundle whose contributions to protein stability and proton pumping are influenced by long-range interactions with the extramembranous carboxyl-terminus.

Monitoring the Conformational Changes of Photoactivated Rhodopsin from Μicroseconds to Seconds by Transient Fluorescence Spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

Physicochemical Characterization of Natural Ionic Microreservoirs: Bacillus subtilis Dormant Spores

The kinetics of proton exchange between dormant spores and aqueous environment was examined by time-resolved micropotentiometry, the method we recently introduced for hydrogel particles of micro- and nanometer diameter (J. Phys. Chem. B 2006, 110, 15107). In this work, the method was applied to the suspensions of dormant Bacillus subtilis spore of different concentrations to show that proton uptake kinetics was a multistep process involving a number of successively approximately 10-fold slower steps of proton penetration into the bulk and their binding to the ionizable groups within different layers of a spore structure. By analyzing the proton equilibrium binding to ionizable groups inside a spore, it was shown that each Bacillus subtilis spore behaves like almost infinite ionic reservoir capable of accumulating billions of protons (N approximately 2 x 10(10) per spore). The obtained pK(a) value of 4.7 for the spores studied is the first quantitative indication on carboxyl groups as the major ionizable groups fixed in a spore matrix. In general, proton equilibrium binding within the spore matrix obeys the fundamental law of the Langmuir isotherm. The proton binding to the ionizable groups slows down the free proton diffusion within a spore, but this effect is substantially weakened by increasing the initial concentration of protons added. On the basis of the diffusion time analysis, it was found that the effective diffusion coefficient for hydrogen ions within the spore core can be up to 3 orders of magnitude lower than that within the coats and cortex. We speculate that the spore inner membrane which separates core from cortex and coats in a dormant spore is a major permeability barrier for protons to penetrate into a lockbox of the genetic information (core).

Ion Concentration of External Solution as a Characteristic of Micro- and Nanogel Ionic Reservoirs

Ion-sensitive hydrogel is regarded as an ionic reservoir, i.e., a system capable of changing the external pH or ionic strength by accumulating or releasing ions. The concept of a hydrogel ionic reservoir was demonstrated for hydrogel particles of three different size ranges: macrogel (1000-6000 microm), microgel (approximately 20-200 microm), and nanogel (approximately 0.2 microm). Ion sensitivity of poly(N-isopropylacrylamide-co-1-vinylimidazole) (PNIPA-VI) microgels with imidazolyl (ionizable) groups was confirmed by the pH dependence of their volume, while nanogels were characterized by dynamic light scattering. On the contrary, the volume of poly(N-isopropylacrylamide) (PNIPA) microgels without ionizable groups was pH independent in the whole range of pH from 10 to 2. Four distinct regions of pH-behavior were observed for PNIPA-VI hydrogel micro- and nanoparticles using potentiometric titration of their suspensions. Time-resolved measurements of ion concentrations in the suspension of hydrogel particles revealed a substantial difference in kinetics of pH equilibration for (i) ion-sensitive hydrogels (PNIPA-VI) vs hydrogels without ionizable groups (PNIPA) and (ii) PNIPA-VI hydrogels of different sizes. On the basis of the experimental observations, a two-step mechanism affecting the kinetics of proton uptake into the hydrogel particles with ionizable groups was proposed: (1) fast binding of ions to the immediate surface of each particle and (2) a slower successive diffusion of bound sites into the next inner layer of polymer network. In accord with the mechanism proposed, a quasi-chemical kinetic model of pH relaxation to equilibrium was developed to fit the experimental data for the time course of proton uptake by macro-, micro-, and nanogels into two exponentials with the characteristic times of tau(1) and tau(2). We believe the same kinetic model will be pertinent to describe phenomenological and molecular mechanisms controlling proton transport in/out bacteria, cells, organelles, drug delivery vehicles, and other natural or artificial multifunctional ionic containers. The approach can be easily extended for the other ions (e.g., Na(+), K(+), and Ca(2+)).

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.

Direct Determination of Proton Uptake Kinetics During the O to E Transition of Cytochrome c Oxidase from P. denitrificans

Direct Determination of Proton Uptake Kinetics During the O to E Transition of Cytochrome c Oxidase from P. denitrificans

Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants.

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.

Proton involvement in the transition from the "peroxy" to the ferryl intermediate of cytochrome c oxidase.

In the absence of any external electron donor, the "peroxy" intermediate of cytochrome c oxidase (CcO-607) is converted to the ferryl form (CcO-580) and subsequently to oxidized enzyme. The rate of conversion of CcO-607 to the CcO-580 form is pH dependent between pH 3.0 and pH 7.6. A plot of the logarithm of the rate constant for this conversion is a linear function of pH with a slope of -0.92, implying the involvement of a single proton in the transition. Upon rapidly lowering the pH from 8.1 to 5.8, the uptake of one proton was observed by direct pH measurement, and the kinetics of proton uptake coincide with the spectral conversion of CcO-607 to CcO-580. We interpret the slow endogenous decay of CcO-607 to CcO-580 to be the result of proton transfer to a deprotonated group generated in the binuclear cavity during CcO-607 formation. This group is not freely accessible to protons from the medium, and its pK(a) is probably higher than 9.0.

Steric interaction between the 9-methyl group of the retinal and tryptophan 182 controls 13-cis to all-trans reisomerization and proton uptake in the bacteriorhodopsin photocycle.

The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.

Protonation and Free Energy Changes Associated with Formation of QBH2 in Native and Glu-L212 .fwdarw. Gln Mutant Reaction Centers from Rhodobacter sphaeroides

Formation of the quinol QBH2 in Glu-L212-->Gln mutant [EQ(L212)] reaction centers (RCs) from Rhodobacter sphaeroides was investigated by measuring the proton uptake (using dyes), UV absorption changes, and free energy changes associated with the two-electron reduction of QB. The advantage of using the EQ(L212) RCs for these studies is that the individual protonation steps can be kinetically resolved and analyzed; conclusions reached regarding the mechanism of formation of QBH2 are expected to apply also to native RCs. The proton uptake by EQ(L212) RCs was strongly biphasic: the fast phase was essentially concomitant with the second electron transfer to QB- (approximately 1 ms at pH 7.5); the slow phase was approximately 2000-fold slower. The rate constant of the slow phase depended on the redox state of the primary quinone QA; for QA- the rate constant was larger (i.e., 8-fold at pH 6.0) than for QA. The electron and proton transfers to QB- in EQ(L212) RCs were modeled with a two-step scheme as follows: (1) fast, QA-QB- + H+(1)-->QA(QBH)-; (2) slow, QA(QBH)- + H+(2)-->QAQBH2, where reaction 1 involves concomitant electron transfer and proton uptake [Paddock, M. L., McPherson, P. H., Feher, G., & Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6803-6807]. The stoichiometry of the fast proton uptake associated with the two-electron reduction of QB varied from 1.1 to 1.4 H+/2e- at pH 6.5-8.5, consistent with the uptake of H+(1) plus an additional fractional proton uptake due to amino acid residues whose pKa values are shifted by interactions with the charge of (QBH)-. The total steady-state proton uptake stoichiometry was 2.0 H+/2e- at pH < or = 7.5, consistent with the formation of the quinol QBH2 (reactions 1 and 2). At pH 8.5, the steady-state proton uptake was 1.6 +/- 0.1 H+/2e-, which is consistent with an apparent pKa for H+(2) of approximately 8.5 [McPherson, P. H., Okamura, M. Y., & Feher, G. (1993) Biochim. Biophys. Acta 1144, 309-324]. The proton uptake kinetics indicate that Glu-L212 is a component of the proton transfer chain for H+(2) that connects reduced QB (buried in the RC protein) to the aqueous solvent as proposed previously [Paddock, M. L., Rongey, S. H., Feher, G., & Okamura, M. Y. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6602-6606].(ABSTRACT TRUNCATED AT 400 WORDS)

Pathway of proton uptake in the bacteriorhodopsin photocycle.

The time courses of chromophore reactions and proton uptake in the second half of the photocycle of the proton pump bacteriorhodopsin (BR) were examined. At pH > 8.5, the kinetics are simplified by the fact that only the M and N intermediates accumulate. The relaxation kinetics after perturbation of M with a second, blue flash confirm that M<==>N equilibration is the only significant cause of the biphasic M decay. With this feature, the analysis of time-resolved difference spectra yields a scheme which contains two sequential N states connected by a nearly unidirectional reaction. The proton uptake from the bulk, as measured with the pH-indicator dye pyranine, occurs during the decay of the first N rather than the recovery of BR. The results thus suggest the model M2(-1)<==>N(-1) + H+ (from the bulk)<==>N(0)-->BR, where the superscripts indicate the protonation state of the protein relative to BR. M2(-1)-->N(-1) is reprotonation of the Schiff base from D96, N(-1) + H+ (from the bulk)-->N(0) is uptake of proton from the cytoplasmic side, and N(0)-->BR represents 13-cis to all-trans reisomerization of the retinal and other relaxations which regenerate the initial state. R227, a residue near D96, affects the deprotonation of D96 more than the subsequent proton uptake. According to the changed [M2(-1)]/[N(-1)] equilibrium in the R227Q protein, interaction between R227 and D96 is responsible for about 1 pH unit of the decrease in the pKa of D96 during the reprotonation of the Schiff base. According to the pH dependencies of the rate constants in the N(-1)<==>N(0) equilibrium in wild-type and R227Q, interaction with R227 lowers the pKa for proton uptake from the bulk by 0.5 pH unit, to 11. We conclude from the proton uptake kinetics that at physiological pH free energy is converted to proton electrochemical potential in this pump not only as protons are released on the extracellular side [Zimányi, L., Váró. G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] but also as protons are taken up on the cytoplasmic side.

A crucial role for Asp L213 in the proton transfer pathway to the secondary quinone of reaction centers from Rhodobacter sphaeroides

The aspartic acid at position 213 of the L-subunit (Asp L213) of reaction centers from Rb. sphaeroides is one of two acidic residues in the binding site of the secondary quinone (Q B). Alteration of Asp L213 to asparagine by site-directed mutagenesis drastically affected the light-induced proton and electron transfer functions leading to formation of quinol (Q BH 2). The first electron transfer was slowed to a half-time in the millisecond time range, but the equilibrium (Q − AQ B ↔ Q AQ − B) was substantially increased in favor of Q B reduction and the pH dependence of the equilibrium was altered. The stabilization of Q − B is suggested to result from the uncharged nature of the substitution, with the implication that Asp L213 is normally ionized and presents an electrostatic restriction to the first electron transfer. The second electron transfer (Q − AQ − B + 2H + ↔ Q AQ BH 2) was even more severely inhibited and was at least 10 4-times slower than the wild type at pH > 6.5, and after only two flashes the RCs were blocked in the Q − AQ − B state. At lower pH some transfer activity was restored, although with a rate still 10 3-times slower than the wild type. The kinetics of the second electron transfer at low pH corresponded exactly to the kinetics of proton uptake. These data are interpreted as implying an essential role for Asp L213 in the proton transfer pathway leading to the formation of QH 2 after the second flash.