Chromatophores, cytochromes

Rhodospririllum rubrum is the most exhaustively studied of the purple nonsulfur bacteria. The photosynthetic apparatus is located in extensive infolded membrane vesicles called chromatophores. Cytochromes C2, cd, and 1)557.5 have all been found to be associated with the chromatophores (371), with C2 being the major component. Several lines of evidence, including fast kinetics following the excitation of the baeteriochlorophyll photocenter by laser flash, have suggested the scheme shown in Fig. 29, where cytochrome Cj is the immediate source of electrons to the electron-... 

The pathways involved in cyclic photophosphorylation in chloroplasts are not yet established. Electrons probably flow from the Fe-S centers Fdx, Fda, or Fdb back to cytochrome b563 or to the PQ pool as is indicated by the dashed line in Fig. 23-18. Cyclic flow around PSII is also possible. The photophosphorylation of inorganic phosphate to pyrophosphate (PP ) occurs in the chromatophores (vesicles derived from fragments of infolded photosynthetic membranes) from Rho-dospirillum rubrum. The PP formed in this way may be used in a variety of energy-requiring reactions in these bacteria.399 An example is formation of NADH by reverse electron transport. 

Fig. 1. Kinetics and standard free energy changes of electron transfer steps in reaction centers isolated from Rb. sphaeroides. In the chromatophore membrane, a c-type cytochrome (Cyt c,) normally reduces before an electron moves from Qa to Qg. The cytochrome oxidation has a time constant of about 20 fis in Rb. sphaeroides. and 0.5 to 2 p in reaction centers of Rp. viridis and Ch. vinosum, which have bound cytochromes. When the reaction center is excited a second time, Ob" is reduced to...

An electron moves from to Qg in about 200 p,s [28-31,51]. Excitation of the reaction center by a second photon sends another electron from P to Q, and then on to Qg with similar kinetics. The fully reduced Qg now probably picks up two protons from the solvent, dissociates from the reaction center as the quinol (QgH2), and is replaced by a fresh molecule of ubiquinone. Electrons from OgH2 return to P" via a Cyt bc complex and a high-potential, c-type cytochrome. This cyclic electron flow drives proton translocation across the chromatophore membrane, and is coupled to the formation of ATP. 

The other two subunits of the Rp. viridis reaction center are more globular in shape [102], The amino-terminal end of the H subunit has a hydrophobic (and presumably transmembrane) helix that runs parallel to the contact region of helices D and E of subunit M. Most of the rest of H forms a large globular domain at the cytoplasmic end of the L-M complex. The cytochrome subunit sits on the relatively flat surface at the periplasmic end of the L-M complex, in agreement with the observation that the cytochromes react with from this side of the membrane in chromatophores or whole cells. The cytochrome also has an internal axis of 2-fold rotational pseudosymmetry, which includes about 1/3 of its amino acid residues. Two of the four hemes lie on one side of this axis, and two on the other. 

Fig. 4. Arrangement of the prosthetic groups in the Rp. viridis. reaction center, redrawn from Ref. 101. Ob is shown at the site identified by Deisenhofcr et al. [102], but the orientation of the quinone in this site is drawn arbitrarily the exact orientation of Ob in the crystal structure has not been described. The four hemes at the top of the figure are in the cytochrome subunit the other components are in the L-M complex. As in Fig. 3, the normal to the chromatophore membrane is approximately vertical and the periplasmic side of the complex is at the top.

The respiratory complexes diffuse laterally in the membrane with diffusion coefficients of 8 X 10 °-2 X 10 cm s [81-83]. Cytochrome c and ubiquinone have been quoted to diffuse at a velocity (10 cm s ) comparable to that of phospholipid molecules [84,85]. However, the bulky isoprenoid side chain of Q may slow down its mobility [86] in chromatophores, which may be compared with mitochondria, a mobility of 10 cm s has been estimated [87]. Overfield and... 

It has become clear in the recent years that electron transfer chains of mitochondria, chloroplasts and some bacteria all contain a cytochrome be complex with very similar structural and functional properties (see Refs. 87, 176-180). Although we focus here on the mitochondrial Complex III, much information has, in particular, come from studies on the bacterial chromatophore system [8,87,176,178]. 

In chromatophores light flash-induced reduction of the b cytochromes (e.g., in the presence of antimycin) is not associated with conservation of energy, as judged from the lack of an electrogenic spectral change in the carotenoids. It is the antimycin-sensitive reoxidation of cytochromes b (through site i ) that appears electrogenic in this sense (see Refs. 87, 276, 277). This contrasts to models (cf.. Fig. 3.9) of electron translocation by the b cytochromes, and the observations that the relative redox poise of the haems is distorted by Aif- [246,278]. However, the position of carotenoids may be such in the membrane that a local field between the b haems is not sensed until it is delocalised. Such a field could also be distorted by a bound SQ molecule. 

In purple photosynthetic bacteria, and specifically in Rps. sphaeroides and Rps. capsulata, three cytochromes of b type have been identified by means of redox titration, in the dark, of isolated chromatophores [116]. They are characterized by midpoint potentials at pH = 7.0 equal to 0.155, 0.050 and -0.090 V (in Rps. sphaeroides)-, the of the 0.050 V species is pH dependent ( — 60 mV per pH unit) [116,117]. The presence of a cytochrome cc in these organisms, interfering spectrally with cytochrome b, makes the situation unclear as far as the existence of cyt. b E j = 0.155 V) is concerned [118]. The two other cytochromes E = 0.050 and — 0.090 V) have also been resolved kinetically in studies on the photosynthetic electron transport and on the basis of their spectral characteristics (band at 561 nm and a spht bands at 558 and 556 nm, respectively these two cytochromes will be referred to as 6-561 and 6-566 in the following) [119]. 

As stated on several occasions in the previous sections, electrons are delivered to bacterial and PSI-RC by electron carriers which can be isolated as water soluble homogeneous proteins, cytochromes of c type or plastocyanine. These carriers represent also the physiological electron acceptors for the 6/Cj complexes. It has been conceived, therefore, that these proteins can act as diffusable redox mediators between the different complexes, which in turn are thought to be laterally and independently mobile in the membrane lipid bilayer [219]. The location of these carriers would be the interface on one side of the asymmetrically arranged coupling membrane, namely towards the periplasmic space in bacteria (corresponding to the internal volume of chromatophores) or the inner lumen of thylakoids. 

The location of cytochrome C2 in the periplasmic space of purple photosynthetic bacteria has been demonstrated directly by its prompt release following the preparation of sphaeroplasts, and by its accessibility to antibodies in these preparations [220]. Cytochromes c are oxidized in single turnover experiments with a biphasic kinetics (<1 2 and 200-400 /is) this pattern has been interpreted as due to the presence in chromatophores of both cyt. Cj and C2, which are oxidized in series [122]. 

Addition of PPj in the dark to a suspension of chromatophores, supplied with Mg ions, causes a reduction of endogenous f>-type cytochrome and a simultaneous... 

Fig. 6.3. Scheme for PP - and ATP-induced oxidation reduction reactions in cytochromes in R. rubrum chromatophores. 

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.,... 

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. 

Fig. 9. (A) EPR spectra of Rp. viridis chromatophores poised at +473 mV (a), +180 mV (b) and -158 mV (c). Two prominent lines at g=3.3 and 3.09 are indicated. The broad band near 260 mT is due to ferricyanide used as a redox mediator. (B) redox titration of the g=3.3 and g=3.09 EPR signals. The dashed line in the low-potential wave of the fip3.3 titration is a one-component fit yielding a midpoint potential of -20 mV. The inset (B, c) shows the shift in field position of the g=3.3 line plotted as a function of redox potential. Data points for titrations in the positive and negative directions are represented by solid and open symbols, respectively. Figure source Nitschke and Rutherford (1989) Tetraheme cytochrome c subunit of Rhodopseudomonas viridis characterized by EPR. Biochemistry 28 3162.

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

The most disappointing loose ends in the Chromatium cytochrome story are the lack of clear-cut roles for either cytochrome cc or flavin-c562. For the latter we can only offer the proposal of Kennel and co-workers (381) that flavin-C662 enhances the rate of reoxidation of the primary photoreductant X when readded to chromatophores depleted of their flavin-C552, and thus may function somewhere in the chain between X and the C662/C668 complex. 

Majima T, Miyake J, Hara M et al. Light-induced electrical responses of dried chromatophore film Effect of the addition of cytochrome c. Thin Solid Films 1989 180 85-88. 

In preparations from Rhodobacter sphaeroides which do not have a bound cytochrome low temperature electron transfer from P to Qa is reversible. This should also be the case in Rdp. viridis when the cytochromes are oxidised. We have therefore investigated the extent of reversible P oxidation at different redox potentials. In an oxidative titration reversible P formation is seen as the low potential haems are oxidised, it remains at a constant level from 50 to 250mV and then increases again as the high potential haems are oxidised and is lost as P is chemically oxidised. The reversible g=2.00 signal had the same line width and saturation characteristics at 100 and 380mV. The same result was obtained in both chromatophores and isolated reaction centres. 

Fig. 1 Effect of various agents on the phosphorylation of the B875 complex in chromatophores. Phosphorylation in the presence of (a) 40)LtM DCCD (N,N -Dicyclohexylcarbodiimide, inhibitor of the membrane-bound ATP-ase), (b) 40/im DCCD, (c) 1/iM antimycin A (an inhibitor of electron flow between cytochrome b and cytochrome cl), (d) 100IM antimycin A, (e) 100/iM DBMIB, (f) 5mM Na-ascorbate (a reductant), (g) 5mM Na-dithionite (a strong reductant) and (h) 5mM potassium ferricyanide (an oxidant and inhibitor of electron transport at the reaction center). All samples except (a) contained 2jL6g/ml venturicidin.

A 100% depletion of RpFi from washed chromatophores could be achieved by sonication in the presence of EDTA and washing the membranes again (Tab.1). The totally depleted chromatophores did not perform any ATPase activity or phosphorylation rate. - Depleted chromatophores recoupled the isolated and purified RpFi-ATPase protein, and thereby restored light-induced H+ uptake (in the presence of cytochrome c) by 90% and photophosphorylation by 75%. NADH-dependent oxidative phosphorylation was reconstituted by 65%. 

FIGURE 2. Acceptor control by phosphory-lating substrates during cytochrome c-induced 02 consumption in chromatophores from / . palustris, - (1)... 

FIGURE 2 (right). Localization of cytochrome be, complex in R. sphaeroides membrane fractions. Membranes were isolated as described in the text and subjected to SDS-polyacrylamide gel electrophoresis each lane received 75 pg of protein. Protein bands were transferred to nitrocellulose and subjected to immunoblotting with a 1/1500 dilution of anti-iron sulfur protein IgG as described in the text. Lane 1, chromatophores lane 2, bc complex lane 3, outer membrane from chemotrophically grown cells lane 4, purified upper pigmented band lane 5, cytoplasmic membrane from chemotrophically grown cells. 

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