A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with alpha-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, -75 and 187 mV, respectively. In addition, two very small contributions to the alpha-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c1 (lambda m (25 degrees C) = 553.5 nm; E'0 = 238 mV) and four cytochromes b (lambda m (25 degrees C) = 558.6, 561.2, 562.1, 566.1 nm and E'0 = -83, 26, 85, -60 mV).     

Stepwise reduction of cytochromes b-562, b-566 and b-558 in rat liver mitochondria.

1. Addition of KCN to aerobic, rotenone-inhibited rat liver mitochondria with out addition of substrate caused reduction of cytochromes b-562 (having an alpha-band at 562 nm at room temperature), c + c1, and a + a3. The effect of KCN on cytochrome b-562 was reversed by pentachlorophenol, though the effect of KCN on cytochromes c+c1 and a+a3 was not reversed by this uncoupler.2. Addition of ATP to aerobic, rat liver mitochondria inhibited with 500 muM KCN under conditions were cytochromes b-562, c+c1 and a+a3 were reduced, caused reduction of cytochrome b-566. The absorbance spectrum of cytochrome b-566 had an alpha-band at 565.5 nm, a beta-band at 538 nm and a gamma-band at 431 nm, but no shoulder around 558 nm at room temperature. 3. Addition of succinate to rotenone-KCN-inhibited and ATP-treated rat liver mitochondria under conditions where cytochromes b-566, b-562, c+c1 and a+a3 were already fully reduced, caused reduction of cytochrome b-558 (having an alpha-band at 558 nm, a beta-band at 527 nm and a gamma-band at 426 nm at room temperature) after exhaustion of molecular oxygen in the reaction medium, without any contribution from a long-wavelength species (cytochrome b-566). 4. It was concluded that the 558-nm band is not a short-wavelength shoulder of cytochrome b-566, but is due to a different species from cytochrome b-566.     

Cytochrome components of plant microsomes.

The electron transport components of the microsomal fraction of cauliflower buds and mung bean hypocotyls were investigated using split-beam and dual wavelength spectrophotometry under a variety of reducing conditions. Cauliflower microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-559.5 [E'0 = +135 +/- 20 mV; lambdamax (reduced minus oxidised) = 559.5, 527 and 429 nm at 23 degrees C], cytochrome b5 [E'0 = -20 +/- 20 mV; lambdamax (reduced minus oxidised) = 556, 526 and 425 nm at 23 degrees C], cytochromes P-450 and P-420. On the basis of binding studies with ethyl isocyanide, degradation of cytochrome P-450 to P-420, redox potential, aniline binding, and relative rates of reduction by NADPH and NADH, it is suggested that the cytochrome P-450 system is analogous to that mammalian microsomes. Other components, reducible only by dithionite, may also be present. Mung bean microsomes were found to contain an ascorbate-reducible component, termed cytochrome b-562 [E'0 = +120 +/- 20 mV; lambdamax (reduced minus oxidised) = 562, 528 and 430 nm at 23 degrees C], cytochrome b5, and a low potential component which was reducible only by sodium dithionite. No cytochrome P-450 or P-420 could be detected. A general method of analysis of the cytochromes was developed and applied to the microsomes from a variety of plant sources. The results indicate that large variations, both in type and amount of components, occur between the microsomes from different plant materials.     

Electron transport and cytochromes in aerobically grown Proteus mirabilis

The function of the cytochromes in electron transport from NADH to oxygen in aerobically grown Proteus mirabilis has been determined. 77K-Spectra of cytoplasmic membrane suspensions, frozen while catalyzing electron transport from NADH to oxygen, in the presence as well as in the absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide, have been recorded. Analysis of these 77K-spectra revealed that cytochrome b-563 (E'0 = +140 mV), cytochrome b-556 (E'0 = +140 mV) [or alternatively cytochrome b-563/556 (E'0 = +140 mV)] and cytochrome b-557 (E'0 = +50 mV) may function in a Q or b-cycle. The function of cytochrome c-549 (E'0 = +75 mV), which seems to be present only in a very low concentration, and cytochrome b-556 (E'0 = -105 mV), which reacts very slowly to the addition of NADH and oxygen, remains unclear. Cytochrome o, the main oxidase of aerobically grown P. mirabilis cells, can not be detected by the methods described above. Only when the reduced form of cytochrome o is liganded with carbon monoxide a specific alpha-band can be detected at 569 nm at 25 degrees C and 565 nm at 77K.     

Purification and properties of cytochrome b-562.5 from Ulva pertusa.

Cytochrome b-562.5 (Ulva pertusa) was extracted from a green alga, U. pertusa, by homogenization of the thalli in phosphate buffer solution. Purification was carried out by acrinol treatment, ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex column chromatographies, and Sephadex gel filtration. Cytochrome b-562.5 has absorption maxima at 562.5 (alpha), 530.5 (beta), 429 (gamma), and 326 nm (delta) in the reduced form and at 537, 415 (gamma), and 275 nm in the oxidized form. The alpha-band of the reduced form is asymmetric with a shoulder at 560 nm, at liquid nitrogen temperature this band splits into two distinct peaks at 562 and 556.5 nm. The absorption maxima of the pyridine ferrohemochrome appear at 556 (alpha), 523 (beta), and 418 nm (gamma). The cytochrome does not combine with carbon monoxide or cyanide. The preparation of the cytochrome shows little peroxidase activity. The cytochrome is oxidized by ferricyanide and reduced by cysteine, ascorbate, and hydrosulfite. Autoxidation of the cytochrome was found to be very slow. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferri-EDTA system to be +0.20 V at pH7.0. The molecular weight of the cytochrome was estimated by Sephadex gel filtration to be 23x10(3).     

Ubiquinol-cytochrome c oxidoreductase of higher plants. Isolation and characterization of the bc1 complex from potato tuber mitochondria.

A procedure is described for isolation of active ubiquinol-cytochrome c oxidoreductase (bc1 complex) from potato tuber mitochondria using dodecyl maltoside extraction and ion exchange chromatography. The same procedure works well with mitochondria from red beet and sweet potato. The potato complex has at least 10 subunits resolvable by gel electrophoresis in the presence of dodecyl sulfate. The fifth subunit carries covalently bound heme. The two largest ("core") subunits either show heterogeneity or include a third subunit. The purified complex contains about 4 mumol of cytochrome c1, 8 mumol of cytochrome b, and 20 mumol of iron/g of protein. The complex is highly delipidated, with 1-6 mol of phospholipid and about 0.2 mol of ubiquinone/mol of cytochrome c1. Nonetheless it catalyzes electron transfer from a short chain ubiquinol analog to equine cytochrome c with a turnover number of 50-170 mol of cytochrome c reduced per mol of cytochrome c1 per s, as compared with approximately 220 in whole mitochondria. The enzymatic activity is stable for weeks at 4 degrees C in phosphate buffer and for months at -20 degrees C in 50% glycerol. The activity is inhibited by antimycin, myxothiazol, and funiculosin. The complex is more resistant to funiculosin and diuron than the beef heart enzyme. The optical difference spectra of the cytochromes were resolved by analysis of full-spectrum redox titrations. The alpha-band absorption maxima are 552 nm (cytochrome c1), 560 nm (cytochrome b-560), and 557.5 + 565.5 nm (cytochrome b-566, which has a split alpha-band). Extinction coefficients appropriate for the potato cytochromes are estimated. Despite the low lipid and ubiquinone content of the purified complex, the midpoint potentials of the cytochromes (257, 51, and -77 mV for cytochromes c1, b-560, and b-566, respectively) are not very different from values reported for whole mitochondria. EPR spectroscopy shows the presence of a Rieske-type iron sulfur center, and the absence of centers associated with succinate and NADH dehydrogenases. The complex shows characteristics associated with a Q-cycle mechanism of redox-driven proton translocation, including two pathways for reduction of b cytochromes by quinols and oxidant-induced reduction of b cytochromes in the presence of antimycin.     

Spectral and potentiometric analysis of cytochromes from Bacillus subtilis

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion-exchange chromatography. They include two terminal oxidases with CO-binding properties and cyanide sensitivity. One of these is an aa3-type cytochrome c oxidase which has characteristic absorption maxima in the reduced-oxidized difference spectrum at 601 nm in the alpha-band and at 443 nm in the Soret band regions. In the alpha-band two separate electron transitions with Em = +205 mV and Em = +335 mV can be discriminated by redox potentiometric titration. The other CO-binding cytochrome c oxidase contains two cytochrome b components with alpha-band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em + +215 mV, whereas cytochrome b559 has an Em = +140 mV. Furthermore a complex consisting of a cytochrome b564 (Em = +140 mV) associated with a cytochrome c554 (Em = +250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical alpha-peak and stains for heme-catalyzed peroxidase activity on SDS-containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for this activity. A cytochrome b559 (Em = +65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid-point potential of Em = +195 mV has been detected.     

The mitochondria of higher mushrooms: the cytochromes beta of the mitochondria of Agaricus campestris (F.) var. bisporus]

Kinetic studies of cytochrome reduction show that only two beta-type cytochromes reducible in the presence of succinate are present in A. campestris mitochondria. At 25 degrees C, the difference spectrum reduced in the presence of antimycin minus oxidized shows the two beta-type cytochrome have a common, asymetric alpha-band at 560 nm which splits into two peaks at 559 and 562 nm at liquid nitrogen temperature (-196 degrees C).     

Oxidation-reduction properties of the cytochrome b found in the plasma-membrane fraction of human neutrophils. A possible oxidase in the respiratory burst.

The oxidation-reduction midpoint potential of the cytochrome b found in the plasma membrane of human neutrophils has been determined at pH 7.0 (Em,7.0) from measurements of absorption spectra at fixed potentials. In both unstimulated and phorbol myristate acetate-stimulated cells Em,7.0 was -245 mV. Changes in pH affected the Em of the cytochrome b, with a slope of approx. 25 mV/pH unit change. The Em,7.0 of the haem group(s) of the membrane-bound myeloperoxidase of human neutrophils was found to be +34 mV. The plasma membranes contained no detectable ubiquinone, and no iron-sulphur compounds were detected by e.p.r. spectroscopy at 5-20 K. No flavins were detected by e.p.r. spectroscopy. The cytochrome b-245 was not reduced by added NADH or NADPH. Dithionite-reduced cytochrome b-245 formed a complex with CO, supplied as a saturated solution, which was dissociated with 26 microseconds illumination from a xenon flash lamp, and the recombination with CO had a half-time of approx. 6 ms. Partly (80%) reduced cytochrome b-245 was oxidized by added air-saturated buffer with a half-time faster than 1 s at 20 degrees C, a resolution limited by mixing time. These results are compatible with cytochrome b-245 acting as an oxidase.     

Do photosynthetic bacteria contain cytochrome c1?

A method is described for characterizing, c-type cytochromes in bacterial membrane preparations according to molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Applied to the photosynthetic bacterium Rhodopseudomonas sphaeroides this technique is used, together with spectroscopic measurements, to demonstrate that a membrane-bound cytochrome c of mol.wt. 30000 is active in photosynthetic electron transport in addition to the well-known soluble cytochrome, cytochrome c2. The membrane cytochrome has a midpoint potential (E'0) at pH 7 of +290 mV, as compared with +360 mV for purified cytochrome c2. Its alpha-band has a peak near 552 nm, as compared with 550 nm for cytochrome c2. Evidence is presented that chromatophores contain roughly equal amounts of the two cytochromes.     

Potentiometric and spectroscopic properties of the cytochrome o complex of Escherichia coli

Cytochrome o purified from cell membranes of Escherichia coli shows two potentiometrically distinct species with midpoint oxidation-reduction potentials of +265 +/- 5 and +140 +/- 15 mV. The component with the higher potential reacted with carbon monoxide and so likely is the oxygen-reacting heme of the cytochrome o complex. It appears to be responsible for the absorption maximum at 564 nm in reduced minus oxidized difference spectra measured at 77 K. The midpoint potential of the other component was sensitive to oxidation by ferricyanide. This latter component had an absorption maximum at about 554 nm. The inhibitor 2-heptyl-4-hydroxyquinoline N-oxide inhibited reoxidation of reduced cytochrome o by oxygen and modified the spectroscopic behaviour of the 564 nm component. The ratio of the heights of the maxima in the alpha-band region of the absorption spectrum differed in cytochrome o prepared from cloned material from that found in cytochrome o from noncloned sources, in spite of the similar polypeptide compositions of the two preparations.     

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.     

Cytochrome b-563 redox changes in intact CO-fixing spinach chloroplasts and in developing pea chloroplasts.

Intact spinach chloroplasts, capable of high rates of photochemical oxygen evolution with CO2 as electron acceptor (120-350 mumol O2 mg chlorophyll-1 h-1) were examined for cytochrome redox changes. The response of the cytochromes in intact chloroplasts to oxidants and reductants appears to be governed by the permeability of the chloroplast envelope. The low potential cytochromes (b-559LP and b-563) were more slowly reduced at 25 degrees C by dithionite than is the case with broken chloroplasts. At 0 degrees C, the reduction of the low potential cytochromes in intactchloroplasts was extremely slow. The chloroplast envelope is impermeable to ferricyanide, slowly permeable to ascorbate and rapidly permeable to reduced dichlorophenolindophenol. Light-induced redox changes of cytochrome b-563 in intact chloroplasts were examined both at 0 degrees and 25 degrees C. A red/far-red antagonism on the redox changes of cytochrome b-563 was observed at 0 degrees C under anaerobic conditions. 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea (DCMU) inhibited the photoreduction of cytochrome b-563 in red light following far-red illumination. The photooxidation of cytochrome b-563 under anaerobic conditions was not influenced by DCMU or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The photoreduction of cytochrome b-563 under aerobic conditions was much less efficient than its photooxidation under anaerobic conditions. Developing pea chloroplasts showed much greater light-induced redox changes of cytochrome b-563 than did intact spinach chloroplasts. Our data are consistent with the view that cytochrome b-563 functions on a cyclic pathway around Photosystem I, but it appears that cyclic flow is sensitive to the relative poising of the redox levels of cytochrome b-563 and the components of the non-cylic pathway.     

Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium.

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

Detection of cytochrome b+50 in membranes of Rhodospirillum rubrum isolated from aerobically and phototrophically grown cells.

1. Dark equilibrium potentiometric titrations were conducted on membranes purified from Rhodospirillum rubrum in an effort to identify b-type cytochrome components reported in other Rhodospirillaceae. In preparations from aerobically grown cells virtually devoid of bacteriochlorophyll a, three components were observed at 560-540 nm. Their oxidation-reduction midpoint potentials assigned by computer-assisted analysis were +195, +50 and -110 mV at pH 7.0; each of these fitted closely to theoretical single-electron equivalent curves. 2. In chromatophores from phototrophically grown carotenoidless mutant G-9, three components were also observed with E0' +190, +50 and -90mV. 3. The alpha-band of the +50mV component exhibited an absorption maximum near 560nm in difference spectra obtained at fixed oxidation-reduction potentials. 4. This component could be demonstrated most readily in purified membrane preparations and may have been obscured in previous studies by residual cytochrome c'. 5. This is the first definitive report of cytochrome b+50 in membranes from Rs. rubrum and aligns this bacterium with other Rhodospirillaceae in which this component functions in light-driven cyclic electron flow.     

Some effects of o-phenanthroline on electron transport in chromatophores from photosynthetic bacteria

The midpoint potentials of the primary electron acceptors in chromatophores from Rhodopseudomonas spheroides and Chromatium have been studied by titrating the laser-induced P605 and cytochrome c oxidations, respectively. Both midpoint potentials are pH dependent (60 mV/pH unit).o-Phenanthroline shifts the midpoint potentials of the primary acceptors, by +40 mV in Rps spheroides and +135 mV in Chromatium. A similar though less extensive change in midpoint potential was observed in the presence of batho-phenanthroline, but not with 8-hydroxyquinoline. The shifted midpoints retain the same dependence on pH.Some of the effects of o-phenanthroline can be explained by assuming that it chelates the reduced form of the primary electron acceptor. This suggests the presence in the primary electron acceptor of a metal chelated by o- and batho-phenanthroline.In Rps spheroides chromatophores o-phenanthroline inhibits the laser- and flash-induced carotenoid shift at all redox potentials, stimulates the laser-induced P605 oxidation at redox potentials between +350 and +420 mV and slows the decay of the laser-induced cytochrome c oxidation below +180 mV. These effects show that o-phenanthroline may have more than one site of action.     

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c₁, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c₁ were 30000 and 275 mV, respectively. The peak of the reduced-minus-oxidized difference spectrum of cytochrome c₁ was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b-c₁ complex in Rps. palustris.     

Characterization and phenotypic control of the cytochrome content of Escherichia coli

1. Electron-transport particles derived from Escherichia coli grown aerobically contain three b-type cytochromes with mid-point oxidation-reduction potentials at pH7 of +260mV, +80mV and -50mV, with n=1 for each. The variation of these values with pH was determined. 2. E. coli develops a different set of b-type cytochromes when grown anaerobically on glycerol with fumarate or nitrate as terminal electron acceptor. Electron-transport particles of fumarate-grown cells contain b-type cytochromes with mid-point potentials at pH7 of +140mV and +250mV (n=1). These two cytochromes are also present in cells grown with nitrate as terminal acceptor, where an additional cytochrome b with a mid-point potential of +10mV (n=1) is developed. 3. The wavelengths of the alpha-absorption-band maxima of the b-type cytochromes at 77K were: (a) for aerobically grown cells, cytochrome b (E(m7) +260mV), 556nm and 563nm, cytochrome b (E(m7) +80mV), 556nm and cytochrome b (E(m7)-50mV), 558nm; (b) for anaerobically grown cells, cytochrome b (E(m7) +250mV), 558nm, cytochrome b (E(m7) +40mV), 555nm and cytochrome b (E(m7) +10mV), 556nm. 4. Cytochrome d was found to have a mid-point potential at pH7 of +280mV (n=1). 5. Cytochrome a(1) was resolved as two components of equal magnitude with mid-point potentials of +260mV and +160mV (n=1). 6. Redox titrations performed in the presence of CO showed that one of the b-type cytochromes in the aerobically grown cultures was reduced, even at the upper limits of our range of electrode potentials (above +400mV). Cytochrome d was also not oxidizable in the presence of CO. Neither of the cytochromes a(1) was affected by the presence of CO.     

The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of Photosystem II

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of −60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of Photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.