The role of the quinone pool in the cyclic electron-transfer chain of Rhodopseudomonas sphaeroides A modified Q-cycle mechanism

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c₂ oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c₁ and c₂, the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0–3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c₁ and c₂, and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 ix oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c₂ oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c₁ and c₂ is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.     

THE ROLE OF THE QUINONE POOL IN THE CYCLIC ELECTRON-TRANSFER CHAIN OF RHODOPSEUDOMONAS SPHAEROIDES: A MODIFIED Q-CYCLE MECHANISM

(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c(2) oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c(1) and c(2), the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c(1) and c(2), and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c(2) oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c(1) and c(2) is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.     

Regulation of Alternative Pathway Activity in Plant Mitochondria : Deviations from Q-Pool Behavior during Oxidation of NADH and Quinols

External NADH and succinate were oxidized at similar rates by soybean (Glycine max) cotyledon and leaf mitochondria when the cytochrome chain was operating, but the rate of NADH oxidation via the alternative oxidase was only half that of succinate. However, measurements of the redox poise of the endogenous quinone pool and reduction of added quinones revealed that external NADH reduced them to the same, or greater, extent than did succinate. A kinetic analysis of the relationship between alternative oxidase activity and the redox state of ubiquinone indicated that the degree of ubiquinone reduction during external NADH oxidation was sufficient to fully engage the alternative oxidase. Measurements of NADH oxidation in the presence of succinate showed that the two substrates competed for cytochrome chain activity but not for alternative oxidase activity. Both reduced Q-1 and duroquinone were readily oxidized by the cytochrome oxidase pathway but only slowly by the alternative oxidase pathway in soybean mitochondria. In mitochondria isolated from the thermogenic spadix of Philodendron selloum, on the other hand, quinol oxidation via the alternative oxidase was relatively rapid; in these mitochondria, external NADH was also oxidized readily by the alternative oxidase. Antibodies raised against alternative oxidase proteins from Sauromatum guttatum cross-reacted with proteins of similar molecular size from soybean mitochondria, indicating similarities between the two alternative oxidases. However, it appears that the organization of the respiratory chain in soybean is different, and we suggest that some segregation of electron transport chain components may exist in mitochondria from nonthermogenic plant tissues.     

Some properties of the redox components of cytochrome c oxidase and their interactions.

The electron paramagnetic resonance (epr) properties of cytochrome c oxidase have been examined with special attention to the effect of added ligands and of interactions between the redox components. The fully oxidized preparations have a very small g6 signal which increases greatly as the redox potential is made more negative, a process exactly paralleling the disappearance of the g3 signal. The potential for half appearance or disappearance (E_m), respectively, is 380 mV at pH 7.0 and 300 mV at pH 8.5. This identifies the changes as accompanying reduction of cytochrome a₃ because the E_m of the “invisible copper” is 340 mV and pH independent. Nitric oxide (NO) binds reduced cytochrome a₃ to form a paramagnetic species. This resulting epr signal is strongly dependent on the redox state of cytochrome a, another expression of heme-heme interaction in cytochrome oxidase. The NO compound is also unique in that under the appropriate conditions three of the four redox components (cytochrome a₃, cytochrome a, and the “visible” copper) are epr active. In potentiometric titrations in the presence of azide the formation of the azide compound responsible for the g2.9 signal appears to require reduction of both cytochrome a₃ and the “invisible copper.” An internal sulfur compound is present which, at alkaline pH values, can bind the heme responsible for the g6 signal and change it to a low-spin sulfur compound with a signal at approximately g2.6. Evidence is also presented for the cytochrome c oxidase in situ being an equilibrium mixture of two different conformational states.     

Stigmatellin induces reduction of iron-sulfur protein in the oxidized cytochrome bc1 complex

Stigmatellin, a Q(P) site inhibitor, inhibits electron transfer from iron-sulfur protein (ISP) to cytochrome c1 in the bc1 complex. Stigmatellin raises the midpoint potential of ISP from 290 mV to 540 mV. The binding of stigmatellin to the fully oxidized complex, oxidized completely by catalytic amounts of cytochrome c oxidase and cytochrome c, results in ISP reduction. The extent of ISP reduction is proportional to the amount of inhibitor used and reaches a maximum when the ratio of inhibitor to enzyme complex reaches unity. A g = 2.005 EPR peak, characteristic of an organic free radical, is also observed when stigmatellin is added to the oxidized complex, and its signal intensity depends on the amount of stigmatellin. Addition of ferricyanide, a strong oxidant, to the oxidized complex also generates a g = 2.005 EPR peak that is oxidant concentration-dependent. Oxygen radicals are generated when stigmatellin is added to the oxidized complex in the absence of the exogenous substrate, ubiquinol. The amount of oxygen radical formed is proportional to the amount of stigmatellin added. Oxygen radicals are not generated when stigmatellin is added to a mutant bc1 complex lacking the Rieske iron-sulfur cluster. Based on these results, it is proposed that ISP becomes a strong oxidant upon stigmatellin binding, extracting electrons from an organic compound, likely an amino acid residue. This results in the reduction of ISP and generation of organic radicals.     

The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus: a comparative study on the redox properties of the metal clusters present in the dinitrogenase components.

The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of -195 mV (appearance) and -30 mV (disappearance), (2) an axial signal (g(parallel) = 2.00, g perpendicular = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the 'low-potential' rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the 'high-potential' rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exhibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: -80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this 'turnover signal' nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.     

Redox and spectroscopic properties of oxidized MoFe protein from Azotobacter vinelandii

The MoFe protein from Azotobacter vinelandii undergoes a six-electron oxidation by various organic dye oxidants with full retention of initial activity. Reduction of the oxidized protein by S2O42- and by controlled potential electrolysis indicates the presence of two reduction regions at -290 and -480 mV, each requiring three electrons for complete reaction. Control of the oxidation conditions provides a means for preparing two distinct MoFe protein species selectively oxidized by three electrons. Selective reduction of the redox region at -290 mV causes development of the EPR signal associated with fully reduced MoFe protein while reduction at -480 mV produces a change in the visible spectrum but has no effect on the EPR signal intensity. Kinetic differences for reduction of the two redox regions indicate that the cofactor region undergoes a more rapid reaction with reductant than the other metal redox sites.     

Half reduction potentials and oxygen affinity of the cytochromes of Pseudomonas carboxydovorans

Abstract The midpoint redox potentials (E'₀) of the cytochromes of Pseudomonas carboxydovorans have been studied by means of coupled spectrum deconvolution and potentiometric analysis. Membranes of cells grown on different substrates (CO; H₂+ CO₂; or pyruvate) contained cytochromes with similar absorption peaks and redox potentials. The cytochromes of the CO-sensitive main electron pathway of the respiratory chain revealed redox potentials in the same range as mitochondrial cytochromes (cytochrome b -555, about −20 mV; cytochrome c and cytochrome a , about +220 mV). For the cytochromes of the CO-insensitive alternative electron pathway, which allows uninhibited growth and respiration in the presence of high concentrations of CO, redox potentials of approx. +50 mV (cytochrome b -558) and −11 to −215 mV (cytochrome b -561) were determined. Cytochrome [ib-561], earlier proposed as the alternative terminal oxidase o in this organism, was shown to possess the lowest half reduction potential of all the cytochromes present in the cells. Measurements of the apparent K _m value for oxygen revealed a low affinity of cytochrome a ( K _m/ 5 υ M O₂) and a very high affinity of the CO-insensitive oxidase ( K _m < 0.5 μ M O₂). The high affinity to oxygen might be responsible for the CO-insensitivity of this unusual cytochrome o .     

Substrate kinetics of the Acanthamoeba castellanii alternative oxidase and the effects of GMP

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K_Mox values were around 4-5 μM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK_Mox values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K_Mox was around 1.2 μM for succinate and around 11 μM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K_Mox. However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K_Mox increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.     

Redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the gav = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate.

The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been investigated through potentiometric titrations. The potentials were determined by EPR spectroscopic quantitation of the mixed valent hydroxylase as a function of added sodium dithionite in the presence of appropriate mediators. The reduction of the oxidized Fe(III).Fe(III) form to the mixed valent Fe(II).Fe(III) form occurs at 48 mV versus NHE while the potential for the formation of the fully reduced Fe(II).Fe(II) species from the mixed valent form was determined to be -135 mV. Addition of the substrate propylene to the hydroxylase did not have a major effect on the reduction potentials. Introduction of the protein B and the reductase components, however, completely inhibited reduction of the hydroxylase at potentials as far negative as -200 mV. Addition of propylene to all three methane monooxygenase components greatly facilitated hydroxylase reduction. Under these conditions, the fully reduced form of the protein was obtained at potentials of greater than 150 mV. This high redox potential indicates that the oxidized form of the protein is highly reactive, as required for methane oxidation. The present results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase.     

A Gene Encoding the Plant-Like Alternative Oxidase is Present in Phytomonas but Absent in Leishmania spp.

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei , trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.     

Redox potentials of the blue copper sites of bilirubin oxidases

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was > 650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.     

Identification of cytochrome a and a3 in yeast cells.

1) Cells of Saccharomyces cerevisiae have been analysed by single and double-bean spectroscopy. Evidence is given for two components of cytochrome c oxidase in the alpha-region of their absorption spectrum. A rapidly reduceable component with a maximum at 600 nm and a slowly reduceable component with a maximum at 604 nm contribute about equal amounts to the total alpha-absorption of cytochrome c oxidase. 2) The component absorbing at 600 nm was identified as the high-potential component with a redox potential of 340 - 355mV, and the 604-nm component as the low-potential component of cytochrome c oxidase with redox potential of 180 - 190 mV. 3) Both components can be characterized by analysing the reduction kinetics in the presence of carbon monoxide. In the presence of saturating concentrations of carbon monoxide, an oxygen pulse leads to a rapid oxidation and subsequent reduction of cytochrome c oxidase, but the rapid reduction phase at 600 nm completely disappears, demonstrating its identity with cytochrome a3, which, being liganded by carbon monoxide in its reduced state, cannot react any more. The component which becomes oxidized and later reduced in the presence of carbon monoxide -- by definition cytochrome a -- has an absorption maximum at 604 nm. 4) The total extinction change at 604 nm in the presence of carbon monoxide is nearly as high as in its absence, but the reduction occurs in two phases and only the second phase, which contributes 50 - 60% to the total absorbance, corresponds in redox potential and kinetic properties to cytochrome a. Because the redox potential of the first reduction phase is very close to that of the low-potential copper atom of cytochrome c oxidase, it is concluded that the apparent increase in the extinction coefficient of cytochrome a in the presence of carbon monoxide is the result of a strong interaction between the ligand fields of cytochrome a and copper, induced by the binding of carbon monoxide to reduced cytochrome a3.     

Redox Properties of Thermus thermophilus ba3: Different Electron-Proton Coupling in Oxygen Reductases?

A comprehensive study of the thermodynamic redox behavior of the hemes of the ba₃ enzyme from Thermus thermophilus, a B-type heme-copper oxygen reductase, is presented. This enzyme, in contrast to those having a single type of heme, allows the B- and A-type hemes to be monitored separately by visible spectroscopy and the reduction potential of each heme to be determined unequivocally. The relative order of the midpoint reduction potentials of each center changed in the pH range from 6 to 8.4, and both hemes present a significant redox-Bohr effect. For instance, at pH 7, the midpoint reduction potentials of the hemes B and A₃ are 213 mV and 285 mV, respectively, whereas at pH 8.4, the order is reversed: 246 mV for heme B and 199 mV for heme A₃. The existence of redox anticooperativity was established by introducing a redox interaction parameter in a model of pairwise interacting redox centers.     

Oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes.

The oxidation-reduction potentials of ferredoxin-NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were -212 mV and -436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately -60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox-linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by -50 mV/pH in the range pH 5-8. The redox potential for the two-electron reduction of ferredoxin-NADP+ reductase was -344 mV at pH 7 (delta Em = -30 mV/pH). In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by +8 mV and E1 was shifted by -25 mV; the shift in potential for the reductase was +4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by -21 mV and E1 shifted by +20 mV; the reductase potential shifted by +2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin-NADP+ reductase from Anabaena variabilis was complexed with flavodoxin from Azotobacter vinelandii. The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.     

ESR studies of cytochrome c3 from Desulfovibrio desulfuricans strain Norway 4: Midpoint potentials of the four haems,and interactions with ferredoxin and colloidal sulphur

ESR spectra were recorded of cytochrome c₃ from Desulfovibrio desulfuricans, strain Norway 4, after poising at different redox potentials in the presence of dye mediators. The spectra of the four different haems were resolved by their different g values and redox potentials. Estimation of the midpoint potentials indicated that there are two higher-potential haems (both −125 ± 15 mV) and two lower-potential haems (−305 and −325 ± 15 mV). The ESR spectrum of the oxidized cytochrome was slightly shifted by interaction with ferredoxin I from D. desulfuricans, Norway strain. Addition of the oxidizing substrate, colloidal sulphur, to the cytochrome c₃ reduced in the presence of hydrogen + hdrogenase, caused a partial reoxidation of the cytochrome, as judged by ESR and spectrophotometry. If redox titrations were carried out in the presence of colloidal sulphur, only the two higher-potential haems could be reduced, with potentials − 110 ± 15 mV. A mechanism of sulphur reduction by an exposed, low-potential haem is discussed. The reaction may involve insoluble S₈ molecules as reaction intermediates.     

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E ⁰s of –30?mV to –100?mV, three to four hemes have E ⁰s around –170?mV, and seven to eight hemes have a lower E ⁰ of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.     

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273–1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.