Reversely-oriented cytochrome b561 in reconstituted vesicles catalyzes transmembrane electron transfer and supports extravesicular dopamine beta-hydroxylase activity

Cytochrome b561 from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b561 can donate electron equivalents directly to cytochrome c. The purified cytochrome b561 was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b561 were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b561 in the vesicle membranes. Pre-treatment of cytochrome b561 with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b561 in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that cytochrome b561 was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. The addition of a soluble form of dopamine beta-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon the addition of ferricyanide as a mediator between cytochrome b561 and dopamine beta-hydroxylase.     

Cytochrome b561 catalyzes transmembrane electron transfer

Purified cytochrome b561 from bovine adrenal medulla chromaffin vesicles has been reconstituted into phosphatidylcholine vesicles by a detergent-dialysis method. When the reconstituted cytochrome-containing vesicles were preloaded with ascorbic acid and cytochrome c was added to the external medium, the internal ascorbic acid was able to reduce the external cytochrome c. This reduction of cytochrome c was dependent on the presence of cytochrome b561 in the membrane and was not due to leakage of ascorbate from the vesicles. These results demonstrate that cytochrome b561 catalyzes a transmembrane electron transfer.     

Selective perturbation of the intravesicular heme center of cytochrome b561 by cysteinyl modification with 4,4'-dithiodipyridine

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with EPR signals at g(z) = 3.69 and 3.14 and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of purified cytochrome b(561) in an oxidized state with a sulfhydryl reagent, 4,4'-dithiodipyridine, caused the introduction of only one 4-thiopyridine group per b(561) molecule at either Cys57 or Cys125. About half of the heme centers of the modified cytochrome were reduced rapidly with ascorbate as found for the untreated sample, but the final reduction level decreased to approximately 65%. EPR spectra of the modified cytochrome showed that a part of the g(z) = 3.14 low-spin EPR species was converted to a new low-spin species with g(z) = 2.94, although a considerable part of the heme center was concomitantly converted to a high-spin g = 6 species. Addition of ascorbate to the modified cytochrome caused the disappearance or significant reduction of the EPR signals at g(z) = 3.69 and 3.14 of low-spin species and at g = 6.0 of the high-spin species, but not for the g(z) approximately 2.94 species. These results suggested that the bound 4-thiopyridone at either Cys57 or Cys125 affected the intravesicular heme center and converted it partially to a non-ascorbate-reducible form. The present observations suggested the importance of the two well-conserved Cys residues near the intravesicular heme center and implied their physiological roles during the electron donation to the monodehydroascorbate radical.     

Rate of transmembrane electron transfer in chromaffin-vesicle ghosts

The chromaffin vesicle of the adrenal medulla contains a transmembrane electron carrier that may provide reducing equivalents for dopamine beta-hydroxylase in vivo. This electron-transfer system can be assayed by trapping ascorbic acid inside resealed membrane vesicles (ghosts), adding an external electron acceptor such as ferricytochrome c or ferricyanide, and following the reduction of these acceptors spectrophotometrically. Cytochrome c reduction is more rapid at high pH and is proportional to the amount of chromaffin-vesicle ghosts, at least at low ghost concentrations. At pH 7.0, ghosts loaded with 100 mM ascorbic acid reduce 60 microM cytochrome c at a rate of 0.035 +/- 0.010 mu equiv min-1 (mg of protein)-1 and 200 microM ferricyanide at a rate of 2.3 +/- 0.3 mu equiv min-1 (mg of protein)-1. The rate of cytochrome c reduction is accelerated to 0.105 +/- 0.021 mu equiv min-1 (mg of protein)-1 when cytochrome c is pretreated with equimolar ferrocyanide. Pretreatment of cytochrome c with ferricyanide also causes a rapid rate of reduction, but only after an initial delay. The ferrocyanide-stimulated rate of cytochrome c reduction is further accelerated by the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), probably because FCCP dissipates the membrane potential generated by electron transfer. These rates of electron transfer are sufficient to account for electron transfer to dopamine beta-hydroxylase in vivo and are consistent with the mediation of electron transfer by cytochrome b-561.     

Functional coupling between enzymes of the chromaffin granule membrane

The reactions of cytochrome b561 with other redox-active components of the adrenal chromaffin granule were examined using optical difference spectroscopy. It was shown that there is no direct electron transfer between the cytochrome and dopamine beta-hydroxylase, but that in the presence of ascorbate, turnover of dopamine beta-hydroxylase causes an oxidation of the cytochrome, which is partially reversed by the action of the mitochondrial NADH:A-. oxidoreductase. Thus, these three proteins may be functionally coupled via ascorbate. A quantitative study of the relationship between the redox state of the cytochrome and the ascorbate radical concentration measured by EPR showed that ascorbate reduces the cytochrome in a one-electron transfer reaction. Generation of a proton electrochemical gradient across the granule membrane causes only a small (20 mV) increase in the cytochrome midpoint potential suggesting the cytochrome is not a proton pump. The data are consistent with a model in which cytochrome b561, by reacting with ascorbate or ascorbate free radical on either side of the granule membrane, could couple the ascorbate-consuming reaction of the dopamine beta-hydroxylase inside the chromaffin granule to the ascorbate-regenerating reaction of the NADH:A-. oxidoreductase on the outer mitochondrial membrane. The H+-ATPase of the granule membrane could both drive the flow of electrons in the direction from cytosol to granule and replenish protons consumed by the turnover of dopamine beta-hydroxylase inside the granule.     

Properties of two distinct heme centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with different midpoint potentials (+150 and +60 mV) and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of oxidized cytochrome b(561) with diethylpyrocarbonate caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of ascorbate during the treatment. Present EPR analyses showed that, upon the treatment, the g(z) = 3.69 heme species was converted to a non-ascorbate-reducible form, although its g(z)-value showed no appreciable change. The treatment had no effect on the other heme (the g(z) = 3.13 species). Raman data indicated that the two heme b centers adopt a six-coordinated low-spin state, in both the reduced and oxidized forms. There was no significant effect of diethylpyrocarbonate-treatment on the Raman spectra of either form, but the reducibility by ascorbate differed significantly between the two hemes upon the treatment. The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the diethylpyrocarbonate-treated cytochrome b(561) when ascorbate was used as a reductant. This observation suggests that ferrocyanide scavenges monodehydroascorbate radicals produced by the univalent oxidation of ascorbate and, thereby, increases both the reduction rate and the final reduction level of the heme center on the intravesicular side of the diethylpyrocarbonate-treated cytochrome. These results further clarify the physiological role of this heme center as the electron donor to the monodehydroascorbate radical.     

Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts

The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate.     

Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid.

Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.     

The structure of cytochrome b561, a secretory vesicle-specific electron transport protein

Cytochrome b561 is a transmembrane electron transport protein that is specific to a subset of secretory vesicles containing catecholamines and amidated peptides. This protein is thought to supply reducing equivalents to the intravesicular enzymes dopamine-beta-hydroxylase and alpha-peptide amidase. We have purified cytochrome b561 from bovine adrenal chromaffin granules by reverse phase chromatography and have determined internal amino acid sequences from peptides. Complementary oligonucleotides were used to isolate two cDNA clones from a bovine brain library. The structure predicted by the sequences of these cDNAs suggests a highly hydrophobic protein of 273 amino acids which spans the membrane six times with little extramembranous sequence. Cytochrome b561 is not homologous to any other cytochrome and thus represents a new class of electron carriers. RNA blotting experiments indicate that cytochrome b561 is expressed in the adrenal medulla and all brain regions of the cow, but not in visceral organs. This result agrees well with the putative function of this unique cytochrome and with the notion that this protein is localized to large dense-core synaptic vesicles.     

Diethyl pyrocarbonate modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence electron donation to monodehydroascorbate radical: identification of the modification sites by mass spectrometric analysis

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. To elucidate the mechanism of the transmembrane electron transfer, effects of the treatment of purified cytochrome b(561) with diethyl pyrocarbonate, a reagent specific for histidyl residues, were examined. We found that when ascorbate was added to the oxidized form of diethyl pyrocarbonate-treated cytochrome b(561), less than half of the heme iron was reduced but with a very slow rate. In contrast, radiolytically generated monodehydroascorbate radical was oxidized rapidly by the reduced form of diethyl pyrocarbonate-modified cytochrome b(561), as observed for untreated cytochrome b(561). These results indicate that the heme center specific for the electron acceptance from ascorbate was perturbed by the modification of amino acid residues nearby. We identified the major modification sites by mass spectrometry as Lys85, His88, and His161, all of which are fully conserved and located on the extravesicular side of cytochrome b(561) in the membranes. We suggest that specific N-carbethoxylation of the histidyl ligands of the heme b at extravesicular side abolishes the electron-accepting ability from ascorbate.     

Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic haem b centre of cytochrome b561: a unique machinery for the biological transmembrane electron transfer

Cytochromes b(561) are a family of transmembrane proteins found in most eukaryotic cells and contain two haem b prosthetic groups per molecule being coordinated with four His residues from four different transmembrane alpha-helices. Although cytochromes b(561) residing in the chromaffin vesicles has long been known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate, newly found members were apparently lacking in the sequence for putative ascorbate-binding site but exhibiting a transmembrane ferrireductase activity. We propose that cytochrome b(561) has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) atom of the axial His residue at the extravesicular haem center, as an initial step of the transmembrane electron transfer. This mechanism utilizes the well-known electrochemistry of ascorbate for a biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants.     

Conversion of soluble dopamine beta-hydroxylase to a membrane binding form

We have shown that purified bovine soluble dopamine beta-hydroxylase can reconstitute onto preformed phosphatidylserine containing vesicles. The binding is dependent on pH and vesicle phosphatidylserine composition but does not require calcium. Reconstitution appears to be irreversible, with the lipid-bound enzyme possessing hydroxylase activity. Additionally, [14C] phosphatidylserine binds to soluble dopamine beta-hydroxylase and remains bound after several detergent washes. Thus the reconstituted soluble form of the enzyme appears to be functionally analogous to the membranous form. Both the reconstitution data and the lipid binding data suggest that multiple phosphatidylserine molecules bind to the soluble hydroxylase. We propose that noncovalently bound phosphatidylserine moieties, which copurify with the membrane bound form of the enzyme, alone are responsible for anchoring membranous dopamine beta-hydroxylase to chromaffin granule and model membranes.     

The 108-kDA peptidylglycine alpha-amidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation

A 43-kDa protein factor that increases the ability of purified bovine peptidylglycine alpha-amidating monooxygenase (PAM)-A and -B to produce alpha-amidated peptides at physiological pH was purified to homogeneity from bovine neurointermediate pituitary. At each step of the purification, the amount of activity correlated with the amount of protein detected on Western blots by antibody to bovine PAM(561-579). In the bovine neurointermediate pituitary the 108-kDa PAM precursor protein is cleaved to form a peptidylglycine alpha-hydroxylating monooxygenase and a peptidyl-alpha-hydroxyglycine alpha-amidating lyase, which function sequentially in the 2-step formation of alpha-amidated peptides.     

Development of a bacterial system for high yield expression of fully functional adrenal cytochrome b561

Adrenal cytochrome b561 (cyt b561) is the prototypical member of an emerging family of proteins that are distributed widely in vertebrate, invertebrate and plant tissues. The adrenal cytochrome is an integral membrane protein with two b-type hemes and six predicted transmembrane helices. Adrenal cyt b561 is involved in catecholamine biosynthesis, shuttling reducing equivalents derived from ascorbate. We have developed an Escherichia coli system for expression, solubilization and purification of the adrenal cytochrome. The spectroscopic and redox properties of the purified recombinant protein expressed in this prokaryotic system confirm that the cytochrome retains a native, fully functional form over a wide pH range. Mass spectral analysis shows that the N-terminal signal peptide is intact. The new bacterial expression system for cyt b561 offers a sixfold improvement in yield and other substantial advantages over existing insect and yeast cell systems for producing the recombinant cytochrome for structure-function studies.     

Cytochrome b561 is fatty acylated and oriented in the chromaffin granule membrane with its carboxyl terminus cytoplasmically exposed

Two polyclonal antibodies were raised to synthetic peptides corresponding to amino acids Ser21-Tyr35 and Lys247-Phe261 of cytochrome b561. These antibodies were used to test the native orientation of the amino and carboxyl termini of this transmembrane electron transport protein. Carboxyl-terminal epitopes were lost when intact chromaffin granules were treated with Pronase. This result indicates that the carboxyl terminus is cytoplasmically exposed and confirms a theoretical prediction obtained from hydropathy plots. Epitopes that were recognized by an amino-terminal antipeptide antibody were not removed under the same conditions. This finding implied that the amino terminus was not proteolytically accessible on the exterior of the granule. The abundance of threonine and serine residues in the amino-terminal region suggested that the amino terminus could be held in the membrane by covalent fatty acylation. Treatment of purified delipidated cytochrome b561 with hydroxylamine resulted in the release of a fatty acid hydroxamate. Sulfhydryl analysis of purified cytochrome b561 showed that all 3 cysteine residues were in the free sulfhydryl form. These observations indicate that cytochrome b561 is covalently fatty acylated and that the lipid is bound through ester linkages of serine or threonine residues.     

Association of cytochrome b5 and cytochrome P-450 reductase with cytochrome P-450 in the membrane of reconstituted vesicles

A protein-protein association of cytochrome P-450 LM2 with NADPH-cytochrome P-450 reductase, with cytochrome b5, and with both proteins was demonstrated in reconstituted phospholipid vesicles by magnetic circular dichroism difference spectra. A 23% decrease in the absolute intensity of the Soret band of the magnetic CD spectrum of cytochrome P-450 was observed when it was reconstituted with reductase. A difference spectrum corresponding to a 7% decrease in absolute intensity was obtained when cytochrome b5 was incorporated into vesicles that already contained cytochrome P-450 and cytochrome P-450 reductase compared to a decrease of 13% in absolute intensity when cytochrome b5 was incorporated into vesicles that contained only cytochrome P-450. The use of the magnetic circular dichroism confirmed that protein-protein associations that have been detected by absorption spectroscopy between purified and detergent-solubilized proteins also exist in membranes. High ionic strength was shown to interrupt direct electron flow from cytochrome P-450 reductase to cytochrome P-450 but not the electron flow from reductase through cytochrome b5 to cytochrome P-450. Upon incorporation of cytochrome b5 into cytochrome P-450- and cytochrome P-450 reductase-containing vesicles, an increase of benzphetamine N-demethylation activity was observed. The magnitude of this increase was numerically identical to the residual activity of the reconstituted vesicles measured in the presence of 0.3 M KCl. It is concluded that there is a requirement for at least one charge pairing for electron transfer from reductase to cytochrome P-450. These observations are combined in a proposed mechanism of coupled reversible association reactions in the membrane.     

A kinetic analysis of electron transport across chromaffin vesicle membranes

Some types of secretory vesicles, such as the chromaffin vesicles of the adrenal medulla, have cytochrome b561 which is believed to mediate the transfer of electrons across the vesicle membrane. To characterize the kinetics of this process, we have examined the rate of electron transfer from ascorbate trapped within chromaffin vesicle ghosts to external ferricyanide. The rate of ferricyanide reduction saturates at high ferricyanide concentrations. The reciprocal of the rate is linearly related to the reciprocal of the ferricyanide concentration. The internal ascorbate concentration affects the y intercept of this double-reciprocal plot but not the slope. These observations and theoretical considerations indicate that the slope is associated with a rate constant k1 for the oxidation of cytochrome b561 by ferricyanide. The intercept is associated with a rate constant k0 for the reduction of cytochrome b561 by internal ascorbate. From k0 and standard reduction potentials, the rate constant k-0 for the reduction of internal semidehydroascorbate by cytochrome b561 can be calculated. Under conditions prevailing in vivo, this rate of semidehydroascorbate reduction appears to be much faster than the expected rate of semidehydroascorbate disproportionation. This supports the hypothesis that cytochrome b561 functions in vivo to reduce intravesicular semidehydroascorbate thereby maintaining intravesicular ascorbic acid.     

Electron transfer across posterior pituitary neurosecretory vesicle membranes

Secretory vesicles from the neurohypophysis have a transmembrane electron carrier very similar to that found in adrenal medullary chromaffin granules. Two different tests show that ascorbic acid contained in the vesicles will reduce an external electron acceptor. First, reduction of cytochrome c or ferricyanide in the medium by a neurosecretory vesicle suspension can be followed spectrophotometrically. Second, the membrane potential (inside positive) generated by electron transfer can be monitored using the membrane potential-sensitive optical probe Oxonol VI. As in chromaffin granules, this electron transfer is probably mediated by cytochrome b561. It may function to regenerate internal ascorbic acid and to provide reducing equivalents needed by the intravesicular amidating enzyme.     

Electron transfer in chromaffin-vesicle ghosts containing peroxidase.

In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome b-561 functioning as the transmembrane electron carrier. If this is true, then the ascorbate-regenerating system should be capable of providing reducing equivalents to any ascorbate-requiring enzyme, not just dopamine beta-monooxygenase. This may be tested using chromaffin-vesicle ghosts in which an exogenous enzyme, horseradish peroxidase, has been trapped. If ascorbate and peroxidase are trapped together within chromaffin-vesicle ghosts, cytochrome b-561 in the vesicle membrane is found in the reduced form. Subsequent addition of H2O2 causes the cytochrome to become partially oxidized. H2O2 does not cause this oxidation if either peroxidase or ascorbate are absent. This argues that the cytochrome is oxidized by semidehydroascorbate, the oxidation product of ascorbate, rather than by H2O2 or peroxidase directly. The semidehydroascorbate must be internal because the ascorbate from which it is formed is sequestered and inaccessible to external ascorbate oxidase. This shows that cytochrome b-561 can transfer electrons to semidehydroascorbate within the vesicles and that the semidehydroascorbate may be generated by any enzyme, not just dopamine beta-monooxygenase.     

Control of cytochrome oxidase activity. A transient spectroscopy study.

The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.