P.L. Dutton

The kinetic and redox potentiometric resolution of the carotenoid shifts in Rhodopseudomonas spheroides chromatophores: Their relationship to electric field alterations in electron transport and energy coupling

Abstract 1. 1. Three principal phases of the carotenoid band shift in Rhodopseudomonas spheroides chromatophores elicited by a single-turnover flash can be resolved both kinetically and potentiometrically. Phase I (complete in μ s) is apparent over the redox potential limits of the light reaction, i.e. the potential range in which reaction centre bacteriochlorophyll is reduced and the primary electron acceptor oxidized before the flash; thus the band shift is consistent with its response to the formation of P + X − . Phase II (about 25% of the amplitude of Phase I) with an approximate 0.15-ms half time is observed if cytochrome c ( E m7.2 + 295 mV) is chemically reduced before the flash and hence may be in response to the photooxidation of cytochrome c and re-reduction of P + . A much slower phase (PHase III) can also be detected at positive potentials. It is enhanced both in extent and formation rate ( t 1 2 > 5 ms to about 1 ms) over the 150 mV potential range in which cytochrome b 155 ( E m7.2 + 155 mV) becomes chemically reduced. Between 50 and 100 mV this phase is approx. 80–100% of the extent of Phase I. Phase III is abolished by antimycin A as are the oxidation of cytochrome b 155 and the re-reduction of photooxidized cytochrome c . All phases are additive. Thus the formation of the carotenoid band shift is in response to pulsed electron transfer events. Further, using multiple, one-turnover flashes, the extent following each flash, and behaviour of the formation the carotenoid band shift can be clearly explained in terms of the electron flow patterns in the chromatophore. 2. 2. The decay of the carotenoid band shift is stimulated by agents such as the uncoupler carbonyl cyanide p -trifluoromethylphenylhydrazone, and the potassium ionophore valinomycin, irrespective of the suspension redox potential. The decay half time, which after five flashes is in the region of 500 ms without addition, can be reduced to c is used to demonstrate that the course of carotenoid band shift decay has no obligate relationship to the redox state or electron transfer events of the electron carriers. 3. 3. The carotenoid band shifts appear to be generated by electrostatic field alterations resulting from oxidation-reduction reactions between adjacent electron transport carriers. Once formed, the retention of the fields is a function of membrane ion permeability. The location of the field-forming reactions, with respect to the kinetically and thermodynamically defined spans of electron transport, and to their topology within the membrane, is discussed.

Engineering protein structure for electron transfer function in photosynthetic reaction centers

A basic relationship is defined that incorporates the three parameters that effectively modulate the rate of intraprotein electron transfer, namely distance, free energy and reorganization energy. This empirically validated relationship is used to explore the minimal requirements for protein-catalyzed conversion of excited electronic states into stable charge separated states, the essence of photosynthesis.

Electric-field dependence of the quantum yield in reaction centers of photosynthetic bacteria

Abstract Multilayer Langmuir-Blodgett films of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been fabricated with partial net orientation. The films showed substantial electrical response under pulsed illumination. From measurements of the light-induced voltage generated across the Langmuir-Blodgett film, we have succeeded in quantitating the electric-field dependence of the quantum yield of charge separation in photosynthesis. The results presented here are compared with our previous determination of the field effect on quantum yield, in which flash-activated charge separation as a function of the applied field was assayed by the extent of bacteriochlorophyll dimer, (BChl)2, oxidation measured optically at 860 nm. The two methods provided consistent dependencies of quantum yield on applied electric field. Analysis of the data reveals that the quantum yield of (BChl)⨥2BPhQ⨪A formation decreases from a value of 0.96 at zero applied field to about 0.75 for a field of 120 mV/nm vectorially directed to hinder light-activated electron transfer. For oppositely applied fields, the quantum yield saturates at unity. The source of the effects is considered to reside in the electric field dependence of the free-energy difference between the energy levels that are involved in the initial charge separation between the (BChl)2 in the first singlet excited state, (BChl)∗2, through the bacteriopheophytin, BPh, to the primary ubiquinone, QA. Possible contributions to the field-induced loss of quantum yield of (BChl)⨥2BPhQ⨪A formation are: (1) a decrease in the free-energy gap between the states (BChl)∗2 and (BChl)⨥2BPh⨪QA, leading to an increased rate of decay via the excited singlet state back to the ground state; (2) a stimulated return from (BChl)⨥2BPh⨪QA directly or via the (BChl)2 triplet state to the ground state and (3) an impeded electron transfer from (BChl)⨥2BPh⨪QA to (BChl)⨥2BPhQ⨪A. These possibilities are discussed. Correlation of the electrical response with measurements of the photo-induced absorbance change allows determination of the projection of the electron-transfer distance on the normal to the plane of the film, which is in good agreement with previous measurements using different techniques.

Reactions of b-cytochromes with ATP and antimycin A in pigeon heart mitochondria.

In pigeon heart mitochondria antimycin A induces a red shift of less than 1 nm in the λmax of ferrocytochrome bK (Em7.0 + 40 mV; λmax 561 nm) but not of ferro-cytochrome bT (Em7.0 - 30 mV; λmax 565 nm). Antimycin A inhibits the ability of ATP to induce a measurable high-potential cytochrome bT. The stoichiometry of these interactions is one antimycin A per cytochrome bK or cytochrome bT. Antimycin A binding to mitochondrial membranes elicits no significant alteration of the oxidation-reduction midpoint potential of either cytochrome bK or T in the resting state. No spectrophotometric alterations were detected in either ferrocytochrome b on addition of ATP.

The triplet state of reaction center bacteriochlorophyll: Determination of a relative quantum yield

The low temperature EPR signal of the excited triplet state of bacteriochlorophyll has been quantitatively studied in reaction centers from Rhodopseudomonas spheroides (carotenoid free R 26 mutant). Using laser flash excitation the light saturation curve of the triplet signal has been compared with that of the free-radical formation due to photooxidation of P870 under identical optical conditions. This comparison shows that the quantum yield of triplet formation is nearly the same as that of the photochemical bleaching of bacteriochlorophyll.

Orientation of the primary quinone of bacterial photosynthetic reaction centers contined in chromatophore and reconstituted membranes

Abstract The orientation of the reaction center bacteriochlorophyll dimer, (BChl) 2 , and primary quinone, Q I , has been studied by EPR in chromatophores of Rhodopseudomonas sphaeroides R26 and Chromatium vinosum and in the reconstituted membrane multilayers of the isolated Rps. sphaeroides reaction center protein. The similarity in the angular dependence of the (BChl) 2 triplet and Q I ⨪Fe 2+ signals in the chromatophore and reconstituted reaction center membrane multilayers indicates that the reaction center is similarly oriented in both native and model membranes. The principle magnetic axes of the (BChl) 2 triplet are found to lie with the x direction approximately parallel to the plane of the membrane surface, and the z and y directions approx. 10–20° away from the plane of the membrane surface and membrane normal, respectively. The Q I ⨪Fe 2+ signals are found to have the g 1.82 component positioned perpendicular to the plane of the membrane surface, with an orthogonal low-field transition (at g 1.68 in Rps. Sphaeroides and at g 1.62 in C. vinosum ) lying parallel to the plane of the membrane surface. The orientation of Q I was determined by the angular dependence of this signal in Fe 2+ -depleted reaction center reconstituted membrane multilayers, and it was found to be situated most likely with the plane of the quinone ring perpendicular to the plane of the membrane surface.

Effect of reduction of the reaction center intermediate upon the picosecond oxidation reaction of the bacteriochlorophyll dimer in Chromatium vinosum and Rhodopseudomonas viridis

The photo-oxidation of the reaction center bacteriochlorophyll dimer or special pair was monitored at 1235 nm in Chromatium vinosum and at 1301 nm in Rhodopseudomonas viridis. In both species, the photo-oxidation was apparently complete within 10 ps after light excitation and proceeded unimpeded at low temperatures regardless of the prior state of reduction of the traditional primary electron acceptor, a quinone-iron complex. Thus the requirement for an intermediary electron carrier (I), previously established by picosecond measurements in Rps. sphaeroides (see ref. 4), is clearly a more general phenomenon. The intermediary carrier, which involves bacteriopheophytin, was examined from the standpoint of its role as the direct electron acceptor from the photo-excited reaction center bacteriochlorophyll dimer. To accomplish this, the extent of light induced bacteriochlorophyll dimer oxidation was measured directly by the picosecond response of the infrared bands and indirectly by EPR assay of the triplet/biradical, as a function of the state of reduction of the I/I⨪ couple (measured by EPR) prior to activation. Two independent methods of obtaining I in a stably reduced form were used: chemical equilibrium reduction, and photochemical reduction. In both cases, the results demonstrated that the intermediary carrier, which we designate I, alone governs the capability for reaction center bacteriochlorophyll photooxidation, and as such I appears to be the immediate and sole electron acceptor from the light excited reaction center bacteriochlorophyll dimer.

Electric field dependence of recombination kinetics in reaction centers of photosynthetic bacteria

Abstract Time-resolved charge recombination has been measured by reflectance/absorption spectroscopic analysis of Langmuir-Blodgett films of reaction centers of the photosynthetic bacterium, Rhodopseudomonas sphaeroides over a wide range of applied electric field strengths. The field dependence of the recombination kinetics has been deduced from the time-course of the reduction of the flash-oxidized bacteriochlorophyll dimer [(BChl) + 2 ] recorded at different applied field strengths. Measurements were performed under two different electric field biasing conditions: a constant bias and a high-frequency bipolar square-wave bias. The additional data obtained from bipolar biasing enabled the use of a new deconvolution method to obtain the field dependence of the rate constants from the experimental curves. The deconvolution shows that the rates for charge recombination from the flash-generated state back to the ground state (BChl) 2 Q A approximate exponential functions of the applied electric field. Correlation of the recombination kinetics data with photoinduced electrical response measurements on films with asymmetric up and down populations of reaction centers reveals that fields opposing charge separation result in faster rates of recombination. Although other possibilities are considered, the main source of the effect is believed to be a result of field-induced changes in the free energy gap between and (BChl) 2 Q A . The results presented here are compared to those obtained in experiments with solubilized reaction centers in which the free energy gap between and (BChl) 2 Q A has been changed by quinone replacement.

The location of redox centers in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex by resonance X-ray diffraction

Abstract The technique of resonance X-ray diffraction (Blasie, J.K. and Stamatoff, J. (1981) Annu. Rev. Biophys. Bioeng. 10, 451–452) utilizing synchrotron radiation was used to determine the locations of the cytochrome c heme iron atom and the photosynthetic reaction center iron atom within the profile of a reconstituted membrane. The accuracy of these determinations was better than ±2 Ȧ. The cytochrome c heme iron atom → reaction center iron atom vector was determined to have a magnitude of approx. 44 Ȧ projected onto the membrane profile and to span most of the lipid hydrocarbon core of the membrane profile. Since the reaction center iron atom interacts magnetically with the primary quinone electron acceptor Q I over a distance of less than 10 Ȧ, the primary light-induced electron-transfer reactions for this system generate the electric charge separation between oxidized cytochrome c + and Fe-Q − I across most (approx. 2 3 ) of the membrane profile including most or all of the lipid hydrocarbon core of the membrane.

The location of redox centers in biological membranes determined by resonance X-ray diffraction. II. Analysis of the resonance diffraction data

In the preceding paper (Stamatoff, J., Eisenberger, P., Blasie, J.K., Pachence, J.M., Tavormina, A., Erecinska, M., Dutton P.L. and Brown, G. (1982) Biochim. Biophys. Acta 679, 177-187), we described the observation of resonance X-ray scattering effects from intrinsic metal atoms associated with redox centers in membrane proteins on the lamellar X-ray diffraction from oriented multilayers of reconstituted membranes. In this paper, we discuss the possible methods of analysis of such data and present the results of our model refinement analysis concerning (a) the location of the cytochrome c heme iron atom in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex and (b) the location of the heme a and a3 iron atoms in the profile structure of a reconstituted membrane containing cytochrome oxidase. The former results are of special importance because they provide a test of the validity of the resonance diffraction data and the methods of analysis, since the location of cytochrome c in the reaction center-cytochrome c membrane profile is known independently of the resonance diffraction experiments.