David M. Tiede

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...

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.

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.

Reconstitution de photochemically active reaction centers in planar phospholipid membranes: Light-induced electrical currents under voltage-clamped conditions

The initial light-activated electron transfer occurring within the photosynthetic bacterial reaction center protein (RC) of Rhodopseudomonas sphaeroides generates a charge separation and a redox potential difference between an oxidized cytochrome c and a reduced quinone. This is summarized in the scheme below in which ferri/ferrocytochrome c is the watersoluble cytochrome c2 that serves as an electron donor to the reaction center; (BChl)2 is a bacteriochlorophyll dimer. BPh is bacteriopheophytin, and Q1 and QU are the reaction center primary and secondary quinones, respectively. The times given are halftimes. See [ 1,2] for recent reviews of the reaction center photochemistry.

Spectrophotometric and voltage clamp characterization of monolayers of bacterial photosynthetic reaction centers

Abstract Bacterial photosynthetic reaction centers from Rhodopseudomonas sphaeroides have been spread on an air/aqueous interface, compressed, and transferred quantitatively to either glass or transparent, tin oxide-coated slides. These assemblies permit the concomitant measurement of both optical and electrical activities to be made on protein films under voltage-clamp conditions. Optical spectra of the monolayer-coated slides reveal characteristic reaction center absorptions. Linear dichroism spectra of the monolayers indicate that the reaction center is aligned on the air/aqueous interface with an angle of inclination which is essentially the same as it is with respect to the membrane plane in vivo. The kinetics of the light-induced absorbance changes of the reaction center in the deposited films are essentially unaltered from those in solution; however, there is some loss in the extent of photochemical activity. Measurement of the light-induced electrical transients shows capacitative charging and discharging currents, which can be readily associated with the reaction center bacteriochlorophyll dimer to ubiquinone electron transfer. The extent of the photochemical activity detected by the voltage-clamp is at best only 10–12% of that measured by optical assay. This suggests that on the air/aqueous interface, the reaction centers must be predominately oriented with opposing directions of electron transfer, having only a slight, variable tendency to align with the ubiquinone directed toward the aqueous phase. In spite of the present shortcomings, these assemblies appear to be uniquely useful to study the effect of clamped potentials on the kinetics and mechanisms of electron transfer.

Conformation and orientation of the protein in the bacterial photosynthetic reaction center

Abstract The protein structure of the Rhodopseudomonas sphaeroides reaction center reconstituted in lipid vesicles was investigated by circular dichroism and polarized infrared spectroscopy. By measuring the infrared dichroism of the amide absorption bands of air-dried oriented membranes (native chromatophores and reconstituted reaction centers), it is possible to estimate the degree of orientation of the polypeptide chains with respect to the bilayer plane. Linear dichroism spectra were investigated from the ultraviolet to the infrared region; measurements of the large linear dichroism of the bacteriochlorophyll and bacteriopheophytin chromophores were used to check the extent of orientation of the air-dried membranes. The major conclusions are: (1) there is a net orientation of the tryptophan heterocycles preferentially in the plane of the membrane. (2) An orientation of the lipids is detected with the polar groups (C=O ester and PO 2 − ) rather parallel to the bilayer plane. (3) The protein of the reaction center is composed to a large extent of α-helix (50 ± 10%) compared to 60 ± 10% in chromatophore membranes. (4) Both the reaction center and the chromatophore membrane contain a small amount of oriented β-structure. (5) The α-helices tend to be aligned along the normal to the membrane. The α-helix axes are tilted at less than 35° in reaction centers and 40° in chromatophores.

Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane.

1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.

Chapter 18 Electrogenic Reactions of the Photochemical Reaction Center and the Ubiquinone-Cytochrome blc2 Oxidoreductase

Publisher Summary This chapter reveals that the photosynthetic bacterium Rhodopseudomonas sphaeroides has a light-driven cyclic electron transport system organized to generate an electric potential and a pH gradient across the cytoplasmic membrane. Although the details are lacking, it is widely accepted that the electrochemical gradients so generated are harnessed for ATP production, NAD + reduction, and solute transport. The results from several approaches together contribute to the view of the RC as a light-activatable redox protein that generates both a redox potential (ΔE h ) and a membrane potential (Δ↓). There is evidence that describes the Q-b/c 2 oxidoreductase as a protein capable of using the ΔE h generated by the RC to drive the formation of further Δ↓ and the translocation of protons across the membrane.

CYTOCHROME-REACTION CENTER-QUINONE INTERACTIONS: MODELS FOR BIOLOGICAL ELECTRON TRANSFER

This paper presents a digest of current knowledge of the redox reactions in photosynthetic bacteria, with the idea of providing some simplifying generalities useful to non-biologists. Hopefully it will also provide some basic information which will promote experiments to improve our understanding of tunneling in biological electron transfer.