Roger C. Prince

Electrochemistry of ubiquinones: Menaquinones and plastoquinones in aprotic solvents

First and second half‐wave reduction potentials of a series of 1,4‐benzo‐ and 1,4‐naphtho‐quinones related to the naturally occurring ubiquinones, plastoquinones and menaquinones are correlated with substituent effects. Notably, E of 2,3‐dimethoxy‐1,4‐benzoquinone is positive of the values for the 2,5‐ and 2,6‐dimethoxy isomers, and of the value for methoxy‐1,4‐benzoquinone. This phenomenon is attributed to steric inhibition of resonance when two methoxy groups occupy adjacent positions, and the significance of this orientation in the ubiquinone series is highlighted.

EPR and optical spectroscopic properites of the electron carrier intermediate between the reaction center bacteriochlorophylls and the primary acceptor in Chromatium vinosum

1. A reaction center-cytochrome c complex has been isolated from Chromatium vinosum which is capable of normal photochemistry and light-activated rapid cytochrome c553 and c555 oxidation, but which has no antenna bacteriochlorophyll. As is found in whole cells, ferrocytochrome c553 is oxidized irreversibly in milliseconds by light at 7 K. 2. Room temperature redox potentiometry in combination with EPR analysis at 7 K, of cytochrome c553 and the reaction center bacteriochlorophyll dimer (BChl)2 absorbing at 883 nm yields identical results to those previously reported using optical analytical techniques at 77 K. It shows directly that two cytochrome c553 hemes are equivalent with respect to the light induced (BChl)2+. At 7 K, only one heme can be rapidly oxidized in the light, commensurate with the electron capacity of the primary acceptor (quinone-iron) being unity. 3. Prior chemical reduction of the quinone-iron followed by illumination at 200K, however, leads to the slow (t1/2 approximately equal to 30 s) oxidation of one cytochrome c553 heme, with what appears to be concommitant reduction of one of the two bacteriophytins (BPh) of the reaction center as shown by bleaching of the 760 nm band, a broad absorbance increase at approx. 650 nm and a bleaching at 543 nm. The 800 nm absorbing bacteriochlorophyll is also involved since there is also bleaching at 595 and 800 nm; at the latter wave-length the remaining unbleached band appears to shift significantly to the blue. No redox changes in the 883 absorbing bacteriochlorophyll dimer are seen during or after illumination under these conditions. The reduced part of the state represents what is considered to be the reduced form of the electron carrier (I) which acts as an intermediate between the bacteriochlorophyll dimer and quinone-iron. The state (oxidized c553/reduced I) relaxes in the dark at 200K in t1/2 approx. 20 min but below 77 K it is trapped on a days time scale. 4. EPR analysis of the state trapped as described above reveals that one heme equivalent of cytochrome becomes oxidized for the generation of the state, a result in agreement with the optical data. Two prominent signals are associated with the trapped state in the g = 2 region, which can be easily resolved with temperature and microwave power saturation: one has a line width of 15 g and is centered at g = 2.003; the other, which is the major signal, is also a radical centered at g = 2.003 but is split by 60 G and behaves as though it were an organic free-radical spin-coupled with another paramagnetic center absorbing at higher magnetic field values; this high field partner could be the iron-quinone of the primary acceptor. The identity of two signals associated with I-. is consistent with the idea that the reduced intermediary carrier is not simply BPh-. but also involves a second radical, perhaps the 800 nm bacteriochlorophylls in the reduced state...

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c2, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation (t 1/2 3-5 microseconds). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (102 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Qz (QzH2) reduces the Rieske Center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c2 reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center. UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from gy 1.90 to gy 1.89, and shifts the Em,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c2, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of HII(+); these may reflect additional sites of action of the inhibitor.

Thermodynamic properties of the reaction center of Rhodopseudomonas viridis. In vivo measurement of the reaction center bacteriochlorophyll-primary acceptor intermediary electron carrier.

The thermodynamic properties of redox components associated with the reaction center of Rhodopseudomonas viridis have been characterized with respect to their midpoint potentials and relationship with protons. In particular a midpoint potential for the intermediary electron carrier acting between the reaction center bacteriochlorophyll and the primary acceptor has been determined. The rationale for this measurement was that the light-induced triplet/biradical EPR signal would not be observed if this intermediate was chemically reduced before activation. The midpoint potential of the intermediary at pH 10.8 is about --400 mV (n=1).

Spectroscopic properties of the intermediary electron carrier in the reaction center of Rhodopseudomonas viridis. Evidence for its interaction with the primary acceptor.

Abstract The spectroscopic properties of the intermediary electron carrier (I), which functions between the bacteriochlorophyll dimer, (BChl) 2 , and the primary acceptor quinone · iron, QFe, have been characterized in Rhodopseudomonas viridis . Optically the reduction of I is accompanied by a bleaching of bands at 545 and 790 nm and a broad absorbance increase around 680 nm which we attribute to the reduction of a bacteriopheophytin, together with apparent blue shifts of the bacteriochlorophyll bands at 830 and possibly at 960 nm. Low temperature electron paramagnetic resonance analysis also reveals complicated changes accompanying the reduction of I. In chromatophores I⨪ is revealed as a broad split signal centered close to g 2.003, which is consistent with I⨪ interacting, via exchange coupling and dipolar effects, with the primary acceptor Q⨪Fe. This is supported by experiments with reaction centers prepared with sodium dodecyl sulfate, which lack the Q⨪Fe g 1.82 signal, and also lack the broad split I⨪ signal; instead, I⨪ is revealed as an approximately 13 gauss wide free radical centered close to g 2.003. Reaction centers prepared using lauryl dimethylamine N -oxide retain most of their Q⨪Fe g 1.82 signal, and in this case I⨪ occurs as a mixture of the two EPR signals described above. However, the optical changes accompanying the reduction of I⨪ are very similar in the two reaction center preparations, so we conclude that there is no direct correlation between the two optical and the two EPR signals of I⨪. Perhaps the simplest explanation of the results is that the two EPR signals reflect the reduced bacteriopheophytin either interacting, or not interacting, with Q⨪Fe, while the optical changes reflect the reduction of bacteriophenophytin, together with secondary, perhaps electrochromic effects on the bacteriochlorophylls of the reaction center. However, we are unable to eliminate completely the possibility that there is also some electron sharing between the reduced bacteriopheophytin and bacteriochlorophyll.

Single and multiple turnover reactions in the ubiquinone-cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroides. The physical chemistry of the major electron donor to cytochrome c2, and its coupled reactions

We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c2 in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferricytochrome with a halftime of 1-2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction. The state of reduction of Z, which may be a quinone.protein complex near the inner (cytochrome c2) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c2, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome rereduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c2 oxido-reductase.

The primary acceptor of bacterial photosynthesis: Its operating midpoint potential?

Abstract 1. 1. The equilibrium oxidation-reduction midpoint potentials of the primary acceptors of the purple photosynthetic bacteria Rhodopseudomonas sphaeroides, Chromatium vinosum and Rhodospirillum rubrum have been determined over a wide range of pH values (pH 4.7–11.0). 2. 2. At physiological values of pH the equilibrium reduction of the primary acceptor involves both an electron and a proton, but at higher values of pH, a p K of the reduced form is titrated so that above the p K only an electron is involved in the reduction. 3. 3. The value of the p K in Rps. sphaeroides is approximately pH 10, while in C . vinosum it is close to pH 8, and in R . rubrum it is close to pH 9. 4. 4. During light-induced electron flow, the reduction of the primary acceptor involves only an electron, implying that the kinetically operational midpoint potential is the equilibrium value measured above the p K of the reduced form. In C. vinosum this is −160 mV, in Rps. sphaeroides −180 mV, and in R. rubrum it is −200 mV.

Further studies on the rieske ironsulfur center in mitochondrial and photosynthetic systems: A pK on the oxidized form

The Rieske iron-sulfur center, characterized by an electron paramagnetic spin resonance spectroscopic signal at g 1.90 when in the reduced form, is a widely distributed component of electron transfer systems, having been found in animal [1], yeast [2], and avian [3] mitochondria, chloroplasts [4], the purple sulfur bacterium Chromatium vinosum [5,6] the purple nonsulfur bacteria Rhodopseudomonas sphaeroides [7] and Rps. capsulata [8] and the green sulfur bacterium Chlorobium limicola f. thiosul[atophilum (D. B. Knaff and R. Malkin, personal communication). In a recent paper in this journal [9] we examined the pH-dependency of the oxidation-reduction midpoint potential of the g 1.90 iron-sulfur center in pigeon heart mitochondria and chromatophores from Rps. sphaeroides and Rps. capsulata, and found that it was independent of pH between approx, pH 6 and pH 8. A similar pH independency over this range was subsequently found for the Rieske center in spinach chloroplasts [4] although in the green sulfur bacterium Chl. limicola f. thiosulfatophilum (D. B. Knaff and R. Malkin, personal communication) it was pH dependent ( 6 0 mV/pH unit) from pH 6.8 to 8.4. More recently [10] we have found that chromatophores are stable to a much wider range of pH than we had previously examined, and this has prompted us to re-examine the pH dependency of the midpoint potential of the Rieske center. The new experiments have revealed a pK on the oxidized form of the iron-sulfur center at pH 8 in both Rps. sphaeroides chromatophores and pigeon heart mitochondria. 2. Materials and methods

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.

The Rieske iron-sulfur center in mitochondrial and photosynthetic systems: Em/pH relationships.

One approach to the elucidation of the mechanisms of energy coupling and electron flow has been the thermodynamic characterisation of the electron and hydrogen carriers involved. A considerable literature has accumulated concerning the half reduction potentials (Em) of components which can be monitored either with optical or electron spin resonance spectroscopy (see [ 1,2] for reviews). However, while the pH dependancy of the half reduction potentials of the cytochromes has been extensively studied in photosynthetic, mitochondrial and bacterial [ 1] systems, the pH/E, relationships of the ironsulfur proteins have not yet received much attention. An important candidate for such an examination is the iron-sulfur protein which, in the reduced state, is responsible for the EPR-detectable g 1.90 signal. This component was first described by Rieske et al. [4] in preparations from beef heart mitochondria, and has since become colloquially known as the ‘Rieske ironsulfur center’. Recent exploratory work indicates that the protein may be ubiquitous in energy conserving organelles; it has been found in animal [3] , yeast [2] and avian [4] mitochondria, chloroplasts (R. Malkin, personal communication), the purple sulfur photosynthetic bacterium Chromatium D [5,6], and in the purple non-sulfur photosynthetic bacteria Rhodopseudomonas spheroides [7] and Rps. capsulata [8]. In all cases, the ‘Rieske centers’ have a half-reduction potential in the 250-3 10 mV redox potential range