High voltage redox properties of cytochrome c oxidase.

In earlier studies evidence was obtained for the existence of both high and low redox potential forms of cytochrome a3 (Hendler et al. 1986. Biophys. J. 49:717-729; Hendler and Sidhu. 1988. Biophys. J. 54:121-133). The current paper describes additional experiments that support this conclusion and then reviews a large number of experimental observations that appear to be consistent with the view that cytochrome a3 displays at least (see Sidhu and Hendler. 1990. Biophys. J. 57:1125-1140) two different forms, which are distinguishable by their redox potentials, spectra, and reactivity with CO.     

Characterization of two low Em forms of cytochrome a3 and their carbon monoxide complexes in mammalian cytochrome c oxidase.

Evidence is presented for the existence of two forms of low-potential cytochrome a3. One appears to be similar to the low-spin form reported by Nicholls, P., and V. Hildebrandt (1978 Biochem. J. 173:65-72) and Wrigglesworth, J. M., J. Elsden, A. Chapman, N. Van der Water, and M. F. Grahn (1988. Biochim. Biophys. Acta. 936:452-464). It has a reduced Soret peak near 428 nm and a prominent alpha peak near 602 nm. This form is seen when the enzyme is either supplemented with lipoprotein or incorporated into a liposomal membrane, preexposed to a voltage greater than 400 mV for at least 30 min, and titrated in the presence of approximately 1 mM K3Fe(CN)6. The other form has a reduced Soret peak near 446 nm, and no prominent alpha peak. The 428-nm form has an Em near 175 mV and forms a CO complex with an Em near 225 mV. The 446-nm form has an Em near 200 mV and forms a CO complex with an Em near 335 mV.     

Direct measurement of the initial and early ratios of proton extrusion to oxygen uptake accompanying cytochrome c oxidation by rat liver mitoplasts.

We have recently described new methods that enable the sharp initiation of a respiratory pulse by photolysis of the CO complex of cytochrome oxidase in a stirred suspension of mitochondria, succinate, O2, and CO (Setty, O. H., R. I. Shrager, B. Bunow, B. Reynafarje, A. L. Lehninger, and R. W. Hendler. 1986. Biophys. J. 50:391-404). Data are collected directly into a microcomputer at 10-ms intervals from fast responding O2 and pH electrodes. These procedures eliminate delays and uncertainties due to mixing times, recorder response, and recovery of the O2 electrode from responding to the injection of O2. Correction procedures were also described for the inherent electrode delays. These procedures revealed an initial burst in medium acidification and a lag in O2 uptake that led to H+/O rates of 20-30 during the first 50 ms and relaxed to "normal" levels by 300 ms. Subsequent changes in [H+] and [O2] followed time courses that appeared to be, but were not strictly, first order. We describe here similar studies in which cytochrome c served as electron donor to site III of rat liver mitoplasts. A qualitatively similar but quantitatively smaller burst in medium acidification and H+/O ratio was seen in these studies. Implications of the previous (Setty et al., 1986) and current studies on defining "mechanistic" H+/O ratios are discussed.     

Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer

A one-of-a-kind high speed optical multichannel spectrometer was designed and built at NIH and described in this journal in 1997 [J.W. Cole, R.W. Hendler, P.D. Smith, H.A. Fredrickson, T.J. Pohida, W.S. Friauf. A high speed optical multichannel analyzer. J Biochem Biophys Methods 1997;35:16–174.]. The most unique aspect of this instrument was the ability to follow an entire time course from a single activation using a single sample. The instrument has been used to study rapid kinetic processes in the photon-driven bacteriorhodopsin photocycle and electron transport from cytochrome c to cytochrome aa₃ and from cytochrome aa₃ to oxygen. The present paper describes a second generation instrument with a number of important enhancements which significantly improve its capabilities for multichannel kinetic studies. An example application is presented in which the kinetics of photon-induced proton flow across the biological membrane is measured simultaneously with the individual steps of the photocycle determined optically. Matching the time constants for the two processes indicates which molecular transformations are associated with major proton movements.     

Potentiometric analysis of Escherichia coli cytochromes in the optical absorbance range of 500 nm to 700 nm.

The oxidation-reduction potentials of Escherichia coli cytochromes have been studied by a recently described technique for automated electrodic potentiometry (Hendler, R.W., Songco, D., and Clem, T.R. (1977) Anal. Chem. 49, 1908-1913; Hendler, R.W. (1977) Anal. Chem. 49, 1914-1918), where entire spectra are recorded at a series of solution potentials. New techniques for resolution of the spectra versus voltage data have been applied. The results indicate that a 1-electron transport chain conducts electrons from substrate to cytochrome d, which is the cytochrome oxidase. Cytochrome d contains several components which appear to increase electron transfer first to a 2-electron stage and then to a 4-electron stage for the final reduction of a molecule of oxygen to 2 molecules of water.     

Comparison of the data, analysis, and results of X-ray absorption studies of cytochrome c oxidase

Differences in the methods of analysis of X-ray absorption data used by Powers et al. [Powers, L., Blumberg, W. E., Chance, B., Barlow, C., Leigh, J., Jr., Smith, J., Yonetani, T., Vik, S., & Peisach, J. (1979) Biochim. Biophys. Acta 547, 520-538; Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] and Scott et al. [Scott, R., Schwartz, J., & Cramer S. (1986) Biochemistry 25, 5546-5555] are clarified. In addition, we compare the X-ray absorption data and results for resting cytochrome c oxidase reported by both groups using the same analysis method and conclude apart from any assumptions that the data are not identical.     

Direct electron transfer of redox proteins at the bare glassy carbon electrode

A simple system is presented for the microscale, direct voltammetry of redox proteins, typically 25 micrograms, in the absence of mediators and/or modifiers. The sample consists of a droplet of anaerobic solution laid onto an oversized disc of nitric-acid-pretreated glassy carbon as the working electrode. Very reproducible, Nernstian responses are obtained with horse heart cytochrome c. The midpoint potential Em (pH 7.0) is dependent on the ionic strength, ranging from $293 mV in 1 mM potassium phosphate to $266 mV in 0.1 M phosphate. At fixed buffer and cytochrome concentrations the magnitude of the voltammetric response is found to be independent of pH over six pH units around neutrality. It is suggested that the response of the present system is not complicated by pH-dependent properties of the electrode surface around physiological pH and, therefore, that the use of this system is practical in biochemically oriented studies. Direct, quasi-reversible responses have also been obtained at pH 7.0 (5 mM phosphate) from Desulfovibrio vulgaris. Hildenborough strain, tetraheme cytochrome c3 (pI = 10.0 at 4 C; 3 X Em = -0.32 mV, Em = -0.26 V), and cytochrome c553 (pI = 9.3; Em = +60 mV), and from Megasphaera elsdenii rubredoxin (pI congruent to 3; Em = -353 mV). The latter protein absorbs onto the glassy carbon surface, thus forming a system with possible applications in the electrochemical study of ferredoxin-linked enzymes.     

Microelectrode of the Thomas type using a liquid membrane electrode

By replacing the glass-based pH electrode (L. R. Pucacco, S. K. Corona, H. R. Jacobson, and N. W. Carter (1986) Anal. Biochem. 153, 251-261) with a liquid membrane-based pH electrode, a relatively easy-to-manufacture modified Thomas electrode has been developed. The liquid membrane-based modified Thomas electrode can be manufactured without the special equipment (forge) and materials (glass) required to make the glass membrane pH microelectrode (L. R. Pucacco and N. W. Carter (1976) Anal. Biochem. 73, 501-512). The sensitivity (57.4 +/- 0.22 mV/pH unit), response time (20.0 +/- 2.67 s), and electrical resistance (3.48 +/- 0.67 X 10(11) ohm) of this electrode are similar to those of the glass-based version.     

Redox properties of membrane-bound b-type cytochromes and a soluble c-type cytochrome of nitrate reductase in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.     

Potentiometric studies on yeast complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

The role of c-type cytochromes in the photosynthetic electron transport pathway of Rhodobacter capsulatus

(1) Short flash excitation of membrane vesicles of a cytochrome-c2-deficient mutant of Rhodobacter capsulatus (strain MT-G4/S4) led to rapid oxidation of a c-type cytochrome. In redox titrations, the photooxidation of c-type cytochrome was attenuated with a midpoint of approx. +360 mV. Vesicles from a control strain, MT1131, gave similar results. These findings are consistent with those of Prince et al. (Prince, R.C., Davidson, E., Haith, L.E. and Daldal, F. (1986) Biochemistry 25, 5208-5214). (2) In anaerobic intact cells the extent of rapid re-reduction of c-type cytochrome oxidised after a flash was less in MT-G/S4 than in MT1131. Cytochrome c reduction in both strains was inhibited by myxothiazol. The myxothiazol-sensitive component of the electrochromic absorbance change in cells indicated that rapid charge separation through the cytochrome bc1 complex was less extensive after a flash in MT-G4/S4 than in MT 1131. (3) In anaerobic intact cells and in chromatophores of Rb. capsulatus strain MT-GS18, a mutant deficient in both cytochrome c1 and cytochrome c2, flash excitation led to the oxidation of c-type cytochrome. Redox titrations and spectra of chromatophores suggested that this is the same cytochrome as was photooxidized in vesicles of MT-G4/S4 and MT1131. This result is in contrast with earlier findings (Prince, R.C. and Daldal, F. (1987) Biochim. Biophys, Acta 894, 370-378) in which it was reported that no photooxidation of c-type cytochrome occurred in the absence of c1 and c2, and argues against the possibility that cytochrome c1 can rapidly and directly donate electrons to the reaction centre. (4) It is proposed that a previously uncharacterized, membrane-bound c-type cytochrome (Em7 approximately +360 mV) is present in Rb-capsulatus MT1131, in the c2-deficient mutant MT-G4/34 and in the c1/c2-deficient mutant MTGS18. This cytochrome and cytochrome c2 are alternative electron donors to the reaction centre in strain MT1131.     

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo.

The equilibrium oxidation-reduction mipoint potential (Em) of isolated Rhodopseudomonas sphaeroides cytochrome c2 exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197-208). However, as with mammalian cytochrome c (Brandt, K.G. Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180-4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the Em of cytochrome c2 before and after the conformational change. The Em of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium Em is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c2 to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent Em from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c2 in vivo involve protons.     

The oxidation-reduction potential of the reaction-centre chlorophyll (P700) in Photosystem I. Evidence for multiple components in electron-paramagnetic-resonance signal 1 at low temperature.

The oxidation-reduction potential of the reaction-centre chlorophyll of Photosystem I (P700) in spinach chloroplasts was determined by using the ability of the reaction centre to photoreduce the bound ferredoxin and to photo-oxidize P700 on illumination at 20K as an indicator of the oxidation state of P700. This procedure shows that P700 is oxidized with Em (pH8.0)(mid-point redox potential at pH8.0)congruent to +375mV. Further oxidation of the chloroplast preparations by high concentrations of K3Fe(CN)6(10mM) in the presence of mediating dyes leads to the appearance of a large radical signal with an apparent Em congruent to +470mVA second, light-inducible, radical also appears over the same potential range. We propose that these signals are due to bulk chlorophyll oxidation and not, as was previously thought [Knaff & Malkin (1973) Arch. Biochem. Biophys. 159, 555-562], to reaction-centre oxidation. A number of optical techniques were used to determine Em of P700. Dual-wavelength spectroscopy (697-720nm) indicates Em congruent to +460-+480mV. The spectrum of the sample during the titration showed a large contribution to the signal by bulk chlorophyll oxidation, in agreement with the electron-paramagnetic-resonance results and those of Ke, Sugahara & Shaw [(1975) Biochim. Biophys. Acta 408, 12-25]. The light-induced absorbance change at 435 nm, usually attributed to P700, showed a potential dependence similar to that of bulk chlorophyll oxidation. Determination of Em of P700 on the basis of the appearance of the P700 signal in oxidized-versus-reduced difference spectra showed Em (pH8.0) congruent to +360mV. Measurements of the effect of potential on the irreversible photo-oxidation of P700 at 77K showed that P700 became oxidized in this potential range. We conclude that the reaction-centre chlorophyll of Photosystem I has Em (pH8.0) congruent to +375mV.     

Axial ligands of chloroplast cytochrome b-559: identification and requirement for a heme-cross-linked polypeptide structure

Optical, resonance Raman, and electron paramagnetic resonance spectroscopies have been used to characterize the ligands and spin state of the chloroplast cytochrome b-559. The protein was isolated from both maize and spinach in a low-potential form. The spectroscopic data indicate that the heme iron in both ferric and ferrous cytochrome b-559 is in its low-spin state and ligated in its fifth and sixth coordination positions by histidine nitrogens. Electron paramagnetic resonance data for the purified spinach cytochrome are in good agreement with those determined by Bergstr?m and V?nng?rd [Bergstr?m, J., & V?nng?rd, T. (1982) Biochim. Biophys. Acta 682, 452-456] for a low-potential membrane-bound form of cytochrome b-559. The g values of high-potential cytochrome b-559 are shifted from those of its low-potential forms; this shift is interpreted as arising from a deviation of the planes of the two axial histidine imidazole rings from a parallel orientation. The model is consistent with the physical data and may also account for the facility with which cytochrome b-559 can be converted between low- and high-potential forms. Recent biochemical and molecular biological data [Widger, W. R., Cramer, W. A., Hermodson, M., Meyer, D., & Gullifor, M. (1984) J. Biol. Chem. 259, 3870-3876; Herrmann, R. G., Alt, J., Schiller, D., Cramer, W. A., & Widger, W. R. (1984) FEBS Lett. 179, 239-244] have shown that two polypeptides, one with 83 residues and a second with 39 residues, most likely constitute the protein of the cytochrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coulometric and potentiometric evaluation of the redox components of cytochrome c oxidase in situ

A new coulometric-potentiometric titration cuvette is described which permits accurate measurements of oxidation-reduction components in membranous systems. This cuvette has been utilized to measure the properties of cytochrome c oxidase in intact membranes of pigeon breast muscle mitochondria. The reducing equivalents accepted and donated by the portion of the respiratory chain with half-reduction potentials greater than 200 mV are equal to those required for the known components (cytochrome a3 and the high-potential copper plus cytochrome a, 'visible copper', cytochrome c1, cytochrome c, and the Rieske iron-sulfur protein). Titrations in the presence of CO show that formation of the reduced cytochrome a3-CO complex requires two reducing equivalents per cytochrome a3 (coulometric titration). Potentiometric titrations indicate (Lindsay, J.G., Owen, C.S. and Wilson, D.F. (1975) Arch. Biochem. Biophys. 169, 492--505) that both cytochromes a3 and the high-potential copper must be reduced in order to form the CO complex (n = 2.0 with a CO concentration-dependent half-reduction potential, Em). By contrast, titrations in the presence of azide show that the Em value of the high-potential copper is unchanged by the presence of azide and thus azide binds with nearly equal affinity whether the copper is reduced or oxidized.     

Analysis of the spectra and redox properties of pure cytochromes aa3.

The findings in the current studies with pure cytochrome aa3 confirm the findings in an accompanying paper pertaining to cytochrome aa3 in mitochondria (Reddy et al., 1985). In both cases, three Nernstian titrations are seen with Em values near 200, 260, 340 mV with n values of 2, 2, and 1. Similarly, the alpha absorption features of the difference spectra in both cases were centered near 602, 605, and 607 mn. The component with Em approximately 200 mV was identified as heme a3 on the basis of experiments conducted in an atmosphere of carbon monoxide, and in both cases, the carbon monoxide-liganded species did not display an elevated Em. In the current studies, unique Soret absorbance features are added to the difference spectra for the three Nernstian transitions. Specifically, absorption peaks at 429, 446, and 448 nm go with the alpha peaks seen respectively at 602, 605, and 607 nm. Evidence was presented to support the hypothesis that the redox state of heme alpha may control the redox potential of heme a3.     

The cytochromes in microsomal fractions of germinating mung beans.

Detailed studies of microsomal cytochromes from mung-bean radicles showed the presence of cytochrome P-420, particularly in dark-grown seedlings, accompanied by smaller quantities of cytochrome P-450. Similar proportions of cytochrome P-420 to cytochrome P-450 were found spectrophotometrically in vivo with whole radicles and hypocotyls. Assayed in vitro, maximum concentrations of both cytochromes were attained after 4 days of growth, before undergoing rapid degradation. Illumination of seedlings stabilized cytochrome P-450 and decreased the amount of cytochrome P-420. Three b cytochromes were present in the microsomal fraction, namely cytochromes b-562.5 (Em + 105 +/- 23 mV), b-560.5 (Em + 49 +/- 13 mV) and b5 (Em - 45 +/- 14 mV), all at pH 7.0. Of the b cytochromes, cytochrome b5 alone undergoes a rapid degradation after day 4, Changes in cytochrome b concentrations were confined to the microsomal fraction: mitochondrial b cytochrome concentrations were unaltered with age. Protohaem degradation (of exogenous methaemalbumin) was detected in microsomal fractions of mung beans. The rates of degradation were highest in extracts of young tissue and declined after day 4. The degradation mechanism and products did not resemble those of mammalian haem oxygenase.     

Na/K pump activity in the new membrane formed at first cleavage in Cynops pyrrhogaster eggs

Resting membrane potentials (Em) increased in the negative direction during first cleavage in Cynops pyrrhogaster eggs whose new membranes formed at first cleavage were exposed to bathing solutions by removing the vitelline envelopes. Em was -11.4 and -87.2 mV at the one- and two-cell stages, respectively. Na/K pump activity contributed to Em at the two-cell stage by about -30 mV. The distribution of Na/K ATPase activity was cytochemically studied by Ernst's method (S. A. Ernst, 1972, J. Histochem. Cytochem. 20, 23-38). The new membrane of the eggs at the two-cell stage showed the pump activity. But the activity was detected neither in the preexisting outer membrane of the eggs at the two-cell stage nor in the membrane at the one-cell stage.     

Heterodimer formation between the antimicrobial peptides magainin 2 and PGLa in lipid bilayers: a cross-linking study

The antimicrobial peptides magainin 2 and PGLa, isolated from the skin of the African clawed frog Xenopus laevis, show marked synergism [Westerhoff, H. V., Zasloff, M., Rosner, J. L., Hendler, R. W., de Waal, A., Vaz Gomes, A., Jongsma, A. P. M., Riethorst, A., and Juretic, D. (1995) Eur. J. Biochem. 228, 257-264]. We suggested previously that these peptides form a potent heterodimer composed of either parallel or antiparallel helices in membranes [Matsuzaki, K., Mitani, Y., Akada, K., Murase, O., Yoneyama, S., Zasloff, M., and Miyajima, K. (1998) Biochemistry 37, 15144-15153]. To detect the putative heterodimer by chemical cross-linking, analogues of magainin 2 and PGLa with a Cys residue at either terminus were synthesized. These cross-linking experiments suggested that both peptides form a parallel heterodimer in membranes composed of phosphatidylglycerol/phosphatidylcholine but not in either buffer or a helix-promoting 2,2,2-trifluoroethanol/buffer mixture. The isolated parallel heterodimers exhibited an order of magnitude higher membrane permeabilization activity compared with the monomeric species, indicating that the observed synergism is due to heterodimer formation.     

Fluorescent probes of electrostatic potential 1 nm from the membrane surface

We measured the electrostatic potential 1 nm from the surface of charged phospholipid bilayer membranes to test the predictions of the Gouy-Chapman theory. Fluorescent probes (anthraniloyl, 5-(dimethylamino)naphthalene-1-sulfonyl, Lucifer yellow) were attached covalently to the sialic acid residue of the ganglioside galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylc eramide (GM1). These fluorescent gangliosides were incorporated into neutral [phosphatidylcholine (PC)] or charged [phosphatidylserine (PS)] phospholipid bilayers, and the fluorescence was quenched with the cations thallium and 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (tempamine). We calculated the electrostatic potential at the chromophore from the quenching ratio using the Boltzmann relation: the average potential was -30 mV for PS bilayers in 0.1 M NaNO3. We assume the chromophore is 1 nm from the surface because X-ray diffraction measurements demonstrate that the sialic acid residue of GM1 is 1 nm from the surface of a PC/GM1 bilayer [McDaniel, R. V., & McIntosh, T. J. (1986) Biophys. J. 49, 94-96]. We also used thallium and tempamine to quench the fluorescence of chromophores located at the surface of the PS membranes; in 0.1 M NaNO3 the average surface potential was -80 mV, which agrees with other measurements. The Gouy-Chapman theory predicts that the potential 1 nm from a membrane with a surface potential of -80 mV is -24 mV; this prediction agrees qualitatively with the experimental results obtained with fluorescent gangliosides.