Glutamate 286 in cytochrome aa3 from Rhodobacter sphaeroides is involved in proton uptake during the reaction of the fully-reduced enzyme with dioxygen

The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.     

The mechanism of proton pumping by cytochrome c oxidasex127e comments

Cytochrome c oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the "peroxy" forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme a during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme a and a3 propionates and are driven to the outside ("pumped") by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P --> F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F' with an alternative route to O.     

Mechanism of inhibition of electron transfer by amino acid replacement K362M in a proton channel of Rhodobacter sphaeroides cytochrome c oxidase

The three-dimensional structure of cytochrome coxidase (COX) reveals two potential input proton channels connecting the redox core of the enzyme with the negatively charged (N-) aqueous phase. These are denoted as the K-channel (for the highly conserved lysine residue, K362 in Rhodobacter sphaeroides COX) and the D-channel (for the highly conserved aspartate gating the channel at the N-side, D132 in R. sphaeroides). In this paper, it is shown that the K362M mutant form of COX from R. sphaeroides, although unable to turnover with dioxygen as electron acceptor, can utilize hydrogen peroxide as an electron acceptor, with either cytochrome c or ferrocyanide as electron donors, with turnover that is close to that of the wild-type enzyme. The peroxidase activity is similar to that of the wild-type oxidase and is coupled to the generation of a membrane potential and to proton pumping. In contrast, no peroxidase activity is revealed in the D-channel mutants of COX, D132N, and E286Q. Reduction by dithionite of heme a3 in the fully oxidized oxidase is severely inhibited in the K362M mutant, but not in the D132N mutant. Apparently, mutations in the D-channel arrest COX turnover by inhibiting proton uptake associated with the proton-pumping peroxidase phase of the COX catalytic cycle. In contrast, the K-channel appears to be dispensable for the peroxidase phase of the catalytic cycle, but is required for the initial reduction of the heme-copper binuclear center in the first half of the catalytic cycle.     

Proton uptake controls electron transfer in cytochrome c oxidase

In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the "peroxy" to "oxoferryl" transition, internal transfer of the fourth electron from CuA to heme a has the same rate as proton uptake from the bulk solution (8,000 s-1). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a CuA ligand, was replaced by leucine, which resulted in an increased redox potential of CuA. During reaction of the reduced mutant enzyme with O2, a proton was taken up at the same rate as in the wild-type enzyme (8,000 s-1), whereas electron transfer from CuA to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from CuA to heme a were blocked, the results from this study show that the CuA --> heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a3-CuB before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.     

Involvement of cytochrome c oxidase subunit III in energy coupling

The role of the conserved acidic residues of subunit III of cytochrome c oxidase (COIII) in energy transduction was investigated. Using a COIII deletion mutant of Paracoccus denitrificans, complemented with a plasmid expressing either the wild type (wt) COIII gene or site-directed mutants of the COIII gene, we measured cytochrome c oxidase-dependent ATP synthesis, respiration, and membrane potential. Cytochrome c oxidase-dependent ATP synthesis was attenuated in nonacidic mutants of either Glu98 (E98A and E98Q), or Asp259 (D259A) but not in the acidic mutant E98D. The rates of respiration in the energy conversion-defective mutants were as high as or higher than that in the wt. The cytochrome c oxidase-induced increment of membrane potential in the nonacidic mutants was similar to or higher than that in the wt. In contrast, when succinate-driven ATP synthesis was mediated solely by ubiquinol oxidase (e.g., in the presence of myxothiazol), the rates of ATP synthesis in the nonacidic mutants were higher than that in the wt. Moreover, myxothiazol, which inhibited succinate respiration as well as ATP synthesis in wt and E98D, stimulated ATP synthesis, while inhibiting succinate respiration, in the nonacidic mutants. These results indicate that the attenuation of energy conversion in these mutants is limited to cytochrome c oxidase and thus suggest that subunit III plays a role in energy conversion by cytochrome c oxidase.     

Role of carboxyl residues surrounding heme of human cytochrome b5 in the electrostatic interaction with NADH-cytochrome b5 reductase

To identify the cytochrome b5 residues responsible for the electrostatic interaction with NADH-cytochrome b5 reductase (b5R), we prepared and characterized the cytochrome b5 mutants in which Glu41, Glu42, Glu63, Asp70, and Glu73 were replaced by Ala, utilizing site-directed mutagenesis and the expression system for cytochrome b5 in Escherichia coli. Apparent Km values of the wild type b5R for Glu42Ala cytochrome b5 and Asp70Ala cytochrome b5 were approximately three-fold and six-fold higher than that for the wild type cytochrome b5, respectively, while the kcat values for those mutants were not remarkably affected. In contrast, Glu41Ala, Glu63Ala, and Glu73Ala cytochrome b5 showed almost the same kinetic properties as the wild type cytochrome b5. Furthermore, kinetic studies on combinations of the cytochrome b5 and b5R mutants suggested the interaction between Glu42 and Asp70 of cytochrome b5 and Lys125 and Lys41 of b5R, respectively, in the reaction.     

Single electron reduction of cytochrome c oxidase compound F: resolution of partial steps by transient spectroscopy

The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.     

Photosynthetic electron transport involved in PxcA-dependent proton extrusion in Synechocystis sp. Strain PCC6803: effect of pxcA inactivation on CO2, HCO3-, and NO3- uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na+-dependent proton extrusion. In the presence of 2, 5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO2 fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO2 fixation is inhibited. At pH 8.0, NO3- uptake activity was very low in the pxcA mutant at low [Na+] (approximately 100 microM). At pH 6.5, the pxcA strain did not take up CO2 or NO3- at low [Na+] and showed very low CO2 uptake activity even at 15 mM Na+. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO2, HCO3-, and NO3- is discussed.     

The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn2+ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.     

The water channel of cytochrome c oxidase: inferences from inhibitor studies

Cytochrome c oxidase couples electron transfer to proton transfer from inside the mitochondrion to the cytosol. Protons pass through a channel; it is closed except when protons are pumped. Electron transfer is also coupled to a water cycle. Water moves into and out of the oxidase during electron transfer, presumably through a channel. The three processes are coupled because of the common dependence on electron transfer. If water and protons had to pass through the same channel for the proton to pass, it might be possible to block the pore by entraining small molecules in the flow. The data in this report indicate that there is a correlation between the ability of a compound to inhibit the oxidase and its size. Formamide and formaldehyde are potent inhibitors. Larger and smaller molecules are poor inhibitors. Formamide introduces an internal block in electron transfer. It is a slow-onset, reversible inhibitor, dependent on turnover to manifest its effects. Vesicular oxidase is less influenced by formamide than is soluble oxidase; formamide must pass a permeability barrier to act. The data are consistent with a proton channel with constrictions at both ends that open to yield a pore of approximately 4 A.     

c-type cytochromes and manganese oxidation in Pseudomonas putida MnB1

Pseudomonas putida MnB1 is an isolate from an Mn oxide-encrusted pipeline that can oxidize Mn(II) to Mn oxides. We used transposon mutagenesis to construct mutants of strain MnB1 that are unable to oxidize manganese, and we characterized some of these mutants. The mutants were divided into three groups: mutants defective in the biogenesis of c-type cytochromes, mutants defective in genes that encode key enzymes of the tricarboxylic acid cycle, and mutants defective in the biosynthesis of tryptophan. The mutants in the first two groups were cytochrome c oxidase negative and did not contain c-type cytochromes. Mn(II) oxidation capability could be recovered in a c-type cytochrome biogenesis-defective mutant by complementation of the mutation.     

Experimental and theoretical characterization of the high-affinity cation-binding site of the purple membrane

Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff base vicinity is proposed. In this model, a Mg2+ cation interacts with one oxygen atom of the side chain of Asp85, with both oxygen atoms of Asp212 and with three water molecules. One of these water molecules is hydrogen bonded to both the nitrogen of the protonated Schiff base and the Asp85 oxygen. It could serve as a shuttle for the Schiff base proton to move to Asp85 in the L-M transition.     

Studies on a stabilisation of ubisemiquinone by Escherichia coli quinol oxidase, cytochrome bo

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome-c-binding site and its attendant CuA prosthetic group with a quinone-binding site. The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one-electron steps requires the stabilisation of semiquinone. We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone. The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5-10. The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5. The two-electron couple has an Em7 of approximately 70 mV. Below pH 9, the pH dependence of the two-electron couple is -60mV/pH, indicative of a 2H+/2e- reaction. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q.- species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q.- radicals. Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q.-bo (QH.bo).     

Magnetic resonance studies on the active site and metal centers of Bradyrhizobium japonicum porphobilinogen synthase

Porphobilinogen synthase (PBGS) is a metalloenzyme which catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen. There are at least four types of PBGS, categorized according to metal ion usage. The PBGS from Bradyrhizobium japonicum requires Mg(II) in catalytic metal site A, has an allosteric Mg(II) in metal site C, and also contains an activating monovalent cation binding site [Petrovich et al. (1996) J. Biol. Chem. 271, 8692-8699]. 13C NMR and Mn(II) EPR have been used to probe the active site and Mg(II) binding sites of this 310 000 dalton protein. The 13C NMR chemical shifts of enzyme-bound product demonstrate that the chemical environment of porphobilinogen bound to B. japonicum PBGS is different from that of PBGS which contains Zn(II) rather than Mg(II) at the active site. Use of Mn(II) in place of Mg(II) broadens the NMR resonances of enzyme-bound porphobilinogen, providing evidence for a direct interaction between MnA and product at the active site. Prior characterization of the enzyme defined conditions in which the divalent cation occupies either the A or the C site. Mimicking these conditions allows Mn(II) EPR observation of either MnC or MnA. The EPR spectrum of MnC is significantly broader and less intense than "free" Mn(II), but relatively featureless. The EPR spectrum of MnA is broader still and more asymmetric than MnC. The EPR data indicate that the coordination spheres of the two metals are different.     

The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides

The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochrome c oxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.     

Dibucaine interacts differently with membrane and protein in cytochrome c oxidase systems

Dibucaine acts as a weak protonophore in cytochrome c oxidase proteoliposomes. At low concentrations in the presence of permeant anions, it stimulates turnover and collapses enzyme-generated pH gradients. At higher concentrations, dibucaine inhibits activity of cytochrome c oxidase in proteoliposomes and the isolated enzyme. It also induces a red shift in the resting spectrum, indicating a change at the binuclear centre. This spectroscopic effect is kinetically biphasic. Dibucaine inhibits steady-state oxidase activity, but not the rate of the red shift in the cytochrome a3 Soret band during turnover. It reacts faster with the partially reduced state than with resting enzyme. The inhibition is kinetically biphasic with a noncompetitive Ki approximately 0.5 mM. Excess dibucaine effects a maximal turnover decline of 80%. At low ionic strength only the total Vmax is affected; tight binding of cytochrome c and turnover at the "tight" site are unaffected. Dibucaine may bind to an anionic site in a hydrophobic pocket, modifying electron transfer from cytochrome a and CuA to cytochrome a3 - CuB and the oxidized spectrum of the latter centre. Stimulation of turnover in cytochrome c oxidase in proteoliposomes is due to a separate membrane-dependent proton translocation catalysed by dibucaine in the presence of permeant anions.     

Cytochrome c oxidase from eucaryotes but not from procaryotes is allosterically inhibited by ATP

The activity of reconstituted cytochrome c oxidase from bovine heart but not from Rhodobacter sphaeroides is allosterically inhibited by intraliposomal ATP, which binds to subunit IV. The activity of cytochrome c oxidase of wild-type yeast and of a subunit VIa-deleted yeast mutant, measured with Tween 20-solubilized mitochondria in the presence of an ATP-regenerating system, was also allosterically inhibited by ATP, indicating the general validity of this mechanism of "respiratory control" in eucaryotic cytochrome c oxidases (Arnold and Kadenbach, Eur. J. Biochem. (1997) 249, 350-354). Deletion of subunit VIa changes the biphysic into monophysic kinetics of the yeast enzyme in the presence of ADP. A tenfold higher amount of horse heart cytochrome c, as compared to yeast cytochrome c, was required to relieve the ATP inhibition of the yeast enzyme.     

Substitution of lysine-362 in a putative proton-conducting channel in the cytochrome c oxidase from Rhodobacter sphaeroides blocks turnover with O2 but not with H2O2

The recently reported X-ray structures of cytochrome oxidase reveal structures that are likely proton-conducting channels. One of these channels, leading from the negative aqueous surface to the heme a3/CuB bimetallic center, contains a lysine as a central element. Previous work has shown that this lysine (K362 in the oxidase from Rhodobacter sphaeroides) is essential for cytochrome c oxidase activity. The data presented demonstrate that the K362M mutant is impeded in the reduction of the heme a3/CuB bimetallic center, probably by interfering with the intramolecular movement of protons. The reduction of the heme-copper center is required prior to the reaction with dioxygen to form the so-called peroxy intermediate (compound P). This block can be by-passed to some extent by the addition of H2O2, which can react with the enzyme without prereduction of the heme-copper center and can then be reduced to water using electrons from cytochrome c. Hence, the K362M mutant, though lacking oxidase activity, exhibits cytochrome c peroxidase activity. Rapid mixing techniques have been used to determine the kinetics of this peroxidase activity at concentrations of H2O2 up to 0.5 M. The Km for peroxide is about 50 mM and the Vmax is 50 electrons s-1, which is considerably slower than the turnover that can be obtained for the oxidase activity of the wild-type enzyme (1200 s-1). The turnover of the mutant oxidase with H2O2 appears to be limited by the rate of reaction of the enzyme with peroxide to form compound P, rather than the rate of reduction of compound P to water by cytochrome c. The data require a reexamination of the proposed roles of the putative proton-conducting channels.     

Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions

The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->Asn and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, both in the presence of Cl- (150 mM) and in its absence (75 mM SO4(2-)). Under both conditions the color of the pigment is blue (lambda max = 615 nm). The time course of the photovoltage kinetics is similar to that of the acid-blue form of wild-type, except that an additional transient charge motion occurs with time constants of 60 microseconds and 1.3 ms, indicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is suggested that this is the group XH, which is responsible for proton release in wild-type. At pH 1, the photovoltage signal of D85N changes upon the addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore, the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pKa of the Schiff base, there is a mixture of "M-like" and "N-like" states. Net proton transport in the same direction as in wild-type is restored in D85N from this N-like state. With the mutant Asp212-->Asn (D212N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire pH range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage experiments indicate that net proton pumping is restored. However, this Cl(-)-induced activation of net charge-transport in D212N is only partial. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chloride-dependent features as those accompanying the acid-blue to acid-purple transition of the wild-type.