Cytochrome photooxidation

The light-induced difference spectrum of Cf. aurantiacus shows that the ratio of P865 to MQ in the reaction center is 1 1. By titrating cytochrome photooxidation, a redox potential of - 50 m V is obtained, which most likely reflects the redox potential of the first, i.e., the earlier, menaquinone acceptor. 

WW Parson (1969) Cytochrome photooxidation in Chromatium chromatophores. Each P870 oxidizes two cytochrome hemes. Biochim Biophys Acta 189 397-403... 

Cytochrome Turnover in Native and Mutant RCs. RCs subjected to continuous illumination in the presence of cyt c and excess Q catalyze photooxidation of cyt c (monitored at 550 nm) coupled to the reduction of quinone according the cycle shown in Fig. 6. Native RCs (either Rb. sphaeroides R-26 or 2.4.1) show a rapid cyt c photooxidation (Fig. 7) with a rate constant of 200 s (at saturating cyt c and Qo concentration and light-intensity). The EQ212 mutant RCs exhibit very different kinetics (Fig. 7). They show a fast (within 3 ms) oxidation of 2.9 0.2 cyt c per RC, followed by a slower phase with a rate constant of 7 2 s". The fast oxidation of 3 cyt c indicates the rapid reduction of DQaQb to DQaQb - The slow phase in the cytochrome photooxidation indicates a block in the turnover of the quinone. The reduced turnover rate is consistent with a block in the proton uptake step(s). 

Fig. 7. Cytochrome photooxidation under continuous illumination using Qq as an acceptor catalyzed by either native (Rb. sphaeroides R-26 or 2.4.1) or mutant EQ212 RCs (pH 8, T = 20"C). The oxidation of cyt c was monitored at 550 nm (see Fig. 6 for model of photochemical cycle). The rapid turnover of cytochrome by native RCs is altered in the mutant to give an initial fast oxidation of 3 cyt c and a subsequent slow turnover. From ref. 13.

The discovery by Knaff and Amon(32) of a light-induced photooxidation at — 189°C requiring short-wavelength light has provided information as to a possible primary electron donor for Sn. The photooxidized substance has been identified as a form of cytochrome b absorbing at 559 nm (cytochrome 559)- Pretreatment of the spinach chloroplasts with ferricyanide to oxidize... 

By H NMR monitoring of the oxidation of benzene oxide-oxepine with dimethyldioxirane (DMDO), a significant by-product, oxepine 4,5-dioxide, was identified <1997CRT1314>. This fact supports the hypothesis that the route from oxepine to muconaldehyde proceeds via oxepine 2,3-oxide with a minor pathway leading to symmetrical oxepine 4,5-oxide. The DMDO oxidations provide model systems for the cytochrome P450-dependent metabolism of benzene and atmospheric photooxidation of benzenoid hydrocarbons. 

Fig. 27. Temperature dependence [9] of the characteristic time, r1/2, of electron transfer from cytochrome c to the photooxidized form of chlorophyll for Chromatium bacteria...

The redox partners of these proteins have yet to be identified, although it has been shown that auracyanins can donate electrons to the membrane-bound cytochrome c-554, which is the direct electron donor for the photooxidized bacterial reaction center P870+ (McManus et al., 1992). However, whether it is their proper in vivo function remains uncertain. The sulfocyanin gene is in the same operon with the components of the respiratory electron transfer chain and, since Su. acidocaladar-ius completely lack c-type cytochromes, it is implicated as a substrate for the CuA-containing terminal oxidase. Interestingly, the occurrence of... 

Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Tollin, G., 1993, Kinetics of photooxidation of soluble cytochromes, hipip, and azurin by the photosynthetic reaction center of the purple phototrophic bacterium Rhodopseudomonas viridis Biochemistry 32 471994726. 

Plant plastocyanins are synthesized in the cytosol as 160-170-ammo acid precursor polypeptides consisting of a 60-70-residue transit peptide followed by a 97 99-amino acid mature protein. The transit peptide imports the precursor plastocyanin molecule across the chloroplast envelope and thylakoid membranes to its final destination in the thylakoid lumen, where it shuttles electrons by accepting them from the membrane bound cytochrome / (cyt /) of the cyt b6/f complex and donating them to the photooxidized reaction center P700-I- of photosystem I. Cyanobacterial plastocyanins use an 30-amino acid leader seqnence for thylakoid membrane translocation. Currently, there are more than 100 plant and cyanobacterial plastocyanin sequences that are available either by direct protein sequencing or deduced from the nucleotide sequences of their genes. 

After its photooxidation, P-700 stays oxidized for more than a few microseconds. It is re-reduced by the soluble copper protein plastocyanin or, in cyanobacteria and some algae, by the soluble cytochrome c-553. The relationship between plastocyanin and P-700 has been mainly studied through kinetic analysis of the P-700 ab-... 

Berthomieu C, Boussac A, Mantele W, Breton J, Nabedryk E. (1992) Molecular changes following oxidoreduction of cytochrome b559 characterized by Fourier transform infrared difference spectroscopy and electron paramagnetic resonance Photooxidation in photosystem II and electrochemistry of isolated cytochrome b559 and iron protoporphyrin IX-bisimidazole model compounds. Biochemistry 31 11460-11471. 

Fig. 8. Absorption changes during photooxidation and dark re-reduction of the primary electron donor P (A) and oxidation of cytochrome c (B) following a brief, intense flash. Figure source Parson and Clayton (Straley, Parson, Mauzerall and Clayton) (1973) Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas sphaeroides. Biochim Biophys Acta. 305 606.

Fig. 3. Absorbance change due to quinone reduction in a reaction-center complex isolated from the carotenoidless mutant of Rb. sphaeroides. The sample contained reduced cytochrome and excess ascorbate as the secondary electron-donor system so that photooxidized P does not accumulate. The presence of excess ascorbate kept the oxidized cytochrome reduced, the net quinone reduction spectrum was obtained. Figure source Clayton (1980) Photosynthesis Physical Mechanisms and Chemical Patterns, p 95. Cambridge University Press.

Before the chemical identity of the secondary electron acceptor and the reaction mechanism involved were known. Parson obtained some useful information indirectly from spectro-kinetic studies using a double-flash arrangement. Parson used a pair of laser flashes spaced a few microseconds apart to excite the chromatophores of Chromatium vinosum and found that while the first flash elicited photooxidation of P870, the second flash did not cause another photooxidation even though the photooxidized P870 " has been re-reduced by the endogenous, c-type cytochrome within -2 /js and presumably ready to undergo another photooxidation, provided there had been electron transfer from Qa Io Qb, i.e.,... 

With the only quinone Qa present, a simple pattern of absorbance changes at 450 nm is seen in a series of flashes [Fig. 3 (A)]. After each flash, the photooxidized P870 is presumably re-reduced by a cytochrome molecule and the absorbance decays to its original level as the reduced Qa delivers its electron to an exogenous acceptor present in the medium. The reaction center is then again ready to undergo another charge separation with the next flash, whereupon the cycle is repeated. 

At temperatures sufficiently low that the cytochromes are unable to transfer electrons rapidly to the photooxidized P840, the oxidized primary donor recombines with the reduced electron acceptor P to produce the spin-polarized triplet state through the radical-pair mechanism ... 

Reduction of the Photooxidized Primary Electron Donor by Cytochromes.182... 

Based on the nature of the cytochromes, there are two kinds of photosynthetic bacterial reaction centers. The first kind, represented by that of Rhodobacter sphaeroides, has no tightly bound cytochromes. For these reaction centers, as shown schematically in Fig. 2, left, the soluble cytochrome C2 serves as the secondary electron donor to the reaction center the RC also accepts electrons from the cytochrome bc complex by way ofCytc2- The rate of electron transfer from cytochrome to the reaction center is sensitive to the ionic strength of the medium. Functionally, cytochrome C2 is positioned in a cyclic electron-transport loop. In Rb. sphaeroides, Rs. rubrum and Rp. capsulata cells, the two molecules of cytochromes C2 per RC are located in the periplasmic space between the cell wall and the cell membrane. When chromatophores are isolated from the cell the otherwise soluble cytochrome C2 become trapped and held by electrostatic forces to the membrane surface at the interface with the inner aqueous phase. These cytochromes electrostatically bound to the membrane can donate electrons to the photooxidized P870 in tens of microseconds at ambient temperatures, but are unable to transfer electrons to P870 at low temperatures. 

The second kind of reaction center, as represented by that of Chromatium vinosum or Rhodopseudo-monas viridis, has a tightly bound c-type cytochrome [see Fig. 2, right]. This so-called reaction center-associated cytochrome is a tetraheme of molecular mass of 40 kDa and structurally quite different from the other known, c-type cytochromes. One of the hemes in this RC-associated, c-type cytochrome also serves as the immediate electron donor to the photooxidized primary donor of the photosynthetic bacteria (either P870 in C. vinosum or P960 in Rp. viridis). The oxidized cytochrome in the tetraheme is in turn reduced by the soluble cytochrome C2. The RC-associated cytochromes are not easily dissociated from the RC, even at high ionic strength. 

The subject matter of this chapter is confined to the role of cytochrome as a secondary electron donor, D, i.e., the interaction with the photooxidized primary electron donor formed during the photochemical charge-separation process in photosynthetic bacteria. Another cytochrome, present essentially as a ubiquinone-cytochrome c oxidoreductase in the cytochrome-6ci complex, is particularly important in energy conservation and the creation of a proton gradient for ATP synthesis in of photosynthetic bacteria. This cytochrome fee, complex, is discussed in Chapter 35 dealing with proton transport. 

We now discuss kinetic evidence that supports the notion that a reduced cytochrome is the direct electron donor to the photooxidized P870. . In subsequent sections we discuss properties and reactions of the RC-associated cytochromes, i.e., those cytochromes that are firmly associated with the reaction centers. The topics to be discussed include the temperature-insensitive electron transfer from the cytochrome to the reaction center and the spatial arrangement of the hemes in the tetraheme cytochrome subunit. 

Fig. 3. Top row formulation of the reaction sequence involved In the photochemical charge separation of the photosynthetic bacterial reaction center. (D is the excitation and charge separation. the electron transfer to the (secondary) acceptors, and the electron donation by a secondary donor, the cytochrome, to the photooxidized primary donor P. Figure adapted from RK Clayton (1980) Photosynthesis. Physical Mechanism and Chemical Patterns, p 91. Cambridge Univ Press.

Fig. 4. Absorbance-change kinetics of photooxidation due to the primary eiectron donor and its decay (re-reduction) [upper paneis] and the oxidation of a c-type cytochrome [iower panels] in C. vinosum (left) and Rp. viridis [right panels]. The C. vinosum sample was poised at a redox potential so that Cyt c555 ("Cyt c422 ) is reduced before flash excitation the ambient redox potential in Rp. viridis was -250 mV, so that only Cyt c5S8 is present in the reduced state before excitation. Figure source left panels (C. vinosum) from Parson (1968) The role of P870 in bacterial photosynthesis. Biochim Biophys Acta 153 254 right panels (Rp. viridis) from Shopes, Levine, Molten and Wraight (1987) Kinetics of oxidation of the bound cytochromes in reaction centers from Rhodopseudomonas viridis. Photosynthesis Res 12 167.

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