Oxidation of cytochrome c 2 by photosynthetic reaction centers of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in vivo. Effect of viscosity on the rate of reaction

In Rhodospirillum rubrum and Rhodopseudomonas sphaeroides it is shown that the oxidation of cytochrome c ₂ involves a diffusion limited process. From analysis of the results it follows that the electron transfer probability must be very low. This is corroborated by in vitro studies using the isolated components.     

The reaction domain on Rhodospirillum rubrum cytochrome c2 and horse cytochrome c for the Rhodospirillum rubrum cytochrome bc1 complex

The interaction of the Rhodospirillum rubrum cytochrome bc1 complex with R. rubrum cytochrome c2 and horse cytochrome c was studied using specific lysine modification and ionic strength dependence methods. In order to define the reaction domain on cytochrome c2, several fractions consisting of mixtures of singly labeled carboxydintrophenyl-cytochrome c2 derivatives were employed. Fraction A consisted of a mixture of derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2, while fractions C1, C2, C3, and C4 were mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The rate of the reaction of fraction A was found to be nearly the same as that of native cytochrome c2. However, the rate constants of fractions C1-C4 were found to be more than 20-fold smaller than that of native cytochrome c2. These results indicate that lysine residues surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. Since the same domain is involved in the reaction with the photosynthetic reaction center, cytochrome c2 must undergo some type of rotational or translational diffusion during electron transport in R. rubrum. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse cytochrome c also involves the heme crevice domain.     

The reaction of Rhodospirillum rubrum cytochrome c2 with iron hexacyanides.

The reaction of Rhodospirillum rubrum cytochrome c2 with the nonphysiological reactants, ferrocyanide and ferricyanide has been investigated as a function of ionic strength, temperature and pH, using both stopped-flow and temperature-jump kinetic methods. The results are consistent with a complex reaction mechanism involving the formation of two intermediate complexes. The site of electron transfer appears to be at the front of the cytochrome c2 molecule near the hem e crevice with interacton of both ferri and ferrocyanide with a positively charged region of the molecule. Comparison of the proposed electron transfer mechanism of cytochrome c2 with ferro-ferricyanide is made with the mechanism proposed based upon structural considerations.     

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.     

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylation-independent and methylation-dependent chemotaxis in Rhodobacter sphaeroides and Rhodospirillum rubrum.

In vivo and in vitro methylation, methanol production assays, and the use of specific antibodies raised against the sensory transducing protein Tar in Escherichia coli all failed to demonstrate the presence of methyl-accepting chemotaxis proteins (MCPs) in the photosynthetic bacterium Rhodobacter sphaeroides, although such proteins did exist in another photosynthetic bacterium, Rhodospirillum rubrum. The range of chemicals to which Rhodobacter sphaeroides responds, the lack of an all-or-none response, and the lack of true repellents indicate an alternative chemosensory pathway. The existence of MCPs in Rhodospirillum rubrum means that the lack of MCPs is not the result of a phototrophic metabolism, but may be connected to the unidirectional flagellar motor of Rhodobacter sphaeroides.     

The electric potential field around cytochrome c and the effect of ionic strength on reaction rates of horse cytochrome c.

1. The electric potential fields around tuna ferri- and ferrocytochrome c were calculated assuming that (i) all of the lysines and arginines are protonated, (ii) all of the glutamic and aspartic acids and the terminal carboxylic acid are dissociated, and (iii) the haem has a net charge of +1e in the oxidized form. 2. Near the haem crevice high values for the potential (greater than +2.5 kT/e) are found. Consequently, electron transfer via the haem edge is favored if the oxidant or reductant is negatively charged. 3. The inhomogeneous distribution of charges leads to a dipole moment of 244 and 238 debye for oxidized and reduced tuna cytochrome c, respectively. Horse cytochrome c has dipole moments of 303 (oxidized) and 286 (reduced) debye. 4. A line through the positive and negative charge centres, the dipole axis, crosses the tuna cytochrome c surface at Ala 83 (positive part) and Lys 99 (negative part). The direction of the dipole axis of horse cytochrome c is very similar. Since the centre of the domain on the cytochrome c surface, which is involved in the binding to cytochrome c oxidase, is found at the beta-carbon of the Phe 82 in horse cytochrome c (Ferguson-Miller, S., Brautigan, D.L. and Margoliash, E. (1978) J. Biol. Chem. 253, 149--159) it is suggested that the direction of the dipole is of physiological importance. 5. The activity coefficients of horse ferri- and ferrocytochrome c were calculated as a function of ionic strength using a formula derived by Kirkwood (Kirkwood, J.G. (1934) J. Chem. Phys. 2, 351--361). 6. Due to the high net charge at pH 7.5 the influence of the dipole moments of horse ferri- and ferrocytochrome c on the respective activity coefficients can be neglected at I less than or equal to 50 mM. 7. Using the Br?nsted relation the effect of ionic strength on reaction rates of horse cytochrome c was calculated. Good agreement is found between theory and experimental results reported in the literature.     

Antigenic sites on cytochrome c2 from Rhodospirillum rubrum.

The antigenic determinants for three monoclonal antibodies against cytochrome c2 from Rhodospirillum rubrum were partially characterized by differential chemical modification of free and antibody-bound cytochrome c2 and by cross-reactivity analysis with different antigens. Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c2. The binding of two antibodies was strongly dependent on the native folding of the antigen. The first antibody bound to a determinant around the exposed heme edge on the 'front side' of the molecule which is not antigenic in mitochondrial cytochrome c2. Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome-c oxidase bound to the front side of cytochrome c. This observation points to a subtle conformational adaptation of the antigen induced by the antibody. The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c2 differs from mitochondrial cytochrome c by an eight-residue insertion. The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c2. Only this antibody cross-reacted with isolated cytochrome-c-derived peptides and with apo-cytochrome c2. A preliminary analysis of the polyclonal immune response of five rats against cytochrome c2 indicates that, unlike in eukaryotic cytochrome c, antigenic determinants are distributed over the whole polypeptide chain of the prokaryotic immunogen.     

Role of specific lysine residues in the reaction of Rhodobacter sphaeroides cytochrome c2 with the cytochrome bc1 complex

The reaction of Rhodobacter sphaeroides cytochrome c2 with the Rb. sphaeroides cytochrome bc1 complex was studied by using singly labeled cytochrome c2 derivatives. Cytochrome c2 was treated with chlorodinitrobenzoic acid to modify lysine amino groups to negatively charged carboxydinitrophenyllysines and separated into eight different fractions by ion-exchange chromatography on a Whatman SE 53 (sulfoxyethyl)cellulose column. Peptide mapping studies indicated that six of these fractions were modified at single lysine amino groups. Each of the derivatives had the same Vmax value as native cytochrome c2 in the steady-state reaction with the Rb. sphaeroides cytochrome bc1 complex. However, the Km values of the cytochrome c2 derivatives modified at lysines 10, 55, 95, 97, 99, and 106 were found to be larger than that of native cytochrome c2 by factors of 6, 2, 3, 32, 13, and 8, respectively. These results indicate that lysines located in the sequence 97-106 on the left side of the heme crevice have the greatest involvement in binding the cytochrome bc1 complex. The involvement of lysine 97 is especially significant because it is located in an extra loop comprising residues 89-98 that is not present in eukaryotic cytochrome c.     

Comparative solvent perturbation of horse heart cytochrome c and Rhodospirillum rubrum cytochrome c2.

The extent of exposure of heme to solvent in horse heart cytochrome c and Rhodospirillum rubrum c2 was investigated to determine whether a correlation exists between the properties of these oxidation-reduction proteins and their heme environments. Solvent perturbation absorption difference spectra were measured using ethylene glycol, glycerol, and sucrose at concentrations between 0 and 30%. Cytochrome c appears to exhibit a somewhat greater extent of heme exposure than cytochrome c2 for both the oxidized and reduced states. These results suggest that the lower oxidation-reduction potential of cytochrome c may in part be due to a greater extent of exposure of the heme. The oxidized state of both proteins appears to exhibit a greater exposure than that of the reduced state which is consistent with a more favorable environment for the charge on the ferric heme coordination center.     

The kinetics of photooxidation of c-type cytochromes by Rhodospirillum rubrum reaction centers.

The kinetics of photooxidation of c-type cytochromes from horse heart, Rhodospirillum rubrum, and Rhodopseudomonas capsulata by purified reaction centers from R. rubrum have been investigated. The kinetic mechanism was found to be complex with a second-order step (complex formation) followed by a rate limiting first-order step. Based on studies of the reaction as a function of pH, ionic strength, and detergent concentration, it appears that the complex formation step is largely electrostatically controlled with only portions of the surfaces of the interacting molecules participating. Further, the first-order process observed at high cytochrome concentration appears to result from solvent reorganization and/or a conformational change following complex formation. Based on data analysis in terms of outersphere electron transfer, it is proposed that another first-order process exists which is not rate limiting and is the electron transfer step. Finally, it was found that the detergent concentration can have a profound effect on both the oxidation-reduction potential of the cytochromes and the kinetics of photooxidation. These results limit the detergent concentration range over which experiments can be conducted and interpreted.     

Proton release upon oxidation of tyrosine in reaction centers from Rhodobacter sphaeroides

Markedly different light-induced protonational changes were measured in two reaction center mutants of Rhodobacter sphaeroides. A quadruple mutant containing alterations, at residues L131, M160, M197, and M210, that elevate the midpoint potential of the bacteriochlorophyll dimer was compared to the Y(M) mutant, which contains these alterations plus a tyrosine at M164 serving as a secondary electron donor [Kálmán et al., Nature 402 (1999) 696]. In the quadruple mutant, a proton uptake of 0.1-0.3 H(+)/reaction center between pH 6 and 10 resulted from formation of the oxidized bacteriochlorophyll donor and reduced primary quinone. In the Y(M) mutant, a maximal proton release of -0.5 H(+)/reaction center at pH 8 was attributed to formation of the tyrosyl radical and modeled using electrostatic and direct proton-releasing mechanisms.     

Characterisation of reaction centers and their phospholipids from Rhodospirillum rubrum.

1. Reaction centers from Rhodospirillum rubrum have been extracted with the zwitterionic detergent lauryl dimethyl amine oxide. Subsequent purification has been achieved by gel filtration and ion-exchange chromatography. The pure reaction centers are composed of three protein subunits (L, M, H), bacteriocholorophyll and bacteriopheophytin in the ratio 2 : 1 and phospholipids. 2. The phospholipid composition has been found to be similar to that of whole chromatophore membrane, except that diphosphatidyl glycerol is present in higher amount in the isolated complex. When the detergent treatment of the chromatophore membrane is done in the presence of NaCl, a lower phospholipid content in isolated reaction centers has been found together with a lower stability in the association among the protein subunits. In this complex, the largest subunit H is easily split off and a LM complex is obtained. It is concluded that the phospholipids play an important role in the stability of reaction center complexes.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Regulation of a cytochrome c2 isoform in wild-type and cytochrome c2 mutant strains of Rhodobacter sphaeroides.

In Rhodobacter sphaeroides, mutations that suppress the photosynthetic deficiency (spd mutations) of strains lacking cytochrome c2 (cyt c2) cause accumulation of a periplasmic cyt c2 isoform that has been designated isocytochrome c2 (isocyt c2). In this study, a new method for purification of both cyt c2 and isocyt c2 is described that uses periplasmic fluid as a starting material. In addition, antiserum to isocyt c2 has been used to demonstrate that all suppressor mutants contain an isocyt c2 of approximately 15 kDa. Western blot analysis indicates that isocyt c2 was present at lower levels in both wild-type and cyt c2 mutants than in spd-containing mutants. Although isocyt c2 is detectable under all growth conditions in wild-type cells, the highest level of isocyt c2 is present under aerobic conditions. Our results demonstrate that spd mutations increase the steady state level of isocyt c2 under photosynthetic conditions. Although the physiological function of isocyt c2 in wild-type cells is not known, we show that a nitrate-regulated protein in Rhodobacter sphaeroides f. sp. denitrificans also reacts with the isocyt c2 antiserum.     

Rhodobacter sphaeroides spd mutations allow cytochrome c2-independent photosynthetic growth.

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2) is a periplasmic redox protein required for photosynthetic electron transfer. cyt c2-deficient mutants created by replacing the gene encoding the apoprotein for cyt c2 (cycA) with a kanamycin resistance cartridge are photosynthetically incompetent. Spontaneous mutations that suppress this photosynthesis deficiency (spd mutants) arise at a frequency of 1 to 10 in 10(7). We analyzed the cytochrome content of several spd mutants spectroscopically and by heme peroxidase assays. These suppressors lacked detectable cyt c2, but they contained a new soluble cytochrome which was designated isocytochrome c2 (isocyt c2) that was not detectable in either cycA+ or cycA mutant cells. When spd mutants were grown photosynthetically, isocyt c2 was present at approximately 20 to 40% of the level of cyt c2 found in photosynthetically grown wild type cells, and it was found in the periplasm with cytochromes c' and c554. These spd mutants also had several other pleiotropic phenotypes. Although photosynthetic growth rates of the spd mutants were comparable to those of wild-type strains at all light intensities tested, they contained elevated levels of B800-850 pigment-protein complexes. Several spd mutants contained detectable amounts of isocyt c2 under aerobic conditions. Finally, heme peroxidase assays indicated that, under anaerobic conditions, the spd mutants may contain another new cytochrome in addition to isocyt c2. These pleiotropic phenotypes, the frequency at which the spd mutants arise, and the fact that a frameshift mutagen is very effective in generating the spd phenotype suggest that some spd mutants contain a mutation in loci which regulate cytochrome synthesis.     

Binding of cytochrome c2 to the isolated reaction center of Rhodospirillum rubrum involves the "backside" of cytochrome c2

Lys 109, Lys 112 and Glu 1 of cytochrome c2 from Rhodospirillum rubrum G-9 are about 4-fold less reactive towards acetic anhydride when cytochrome c2 is bound to the isolated photosynthetic reaction center from the same organism. The three shielded residues are clustered together on the "backside" of cytochrome c2. This contrasts with mitochondrial cytochrome c where "frontside" lysines are protected by different physiological electron transfer partners.     

High-resolution 1H- and 15N-NMR studies of Rhodospirillum rubrum cytochrome c2.

Rhodospirillum rubrum cytochrome c2 was uniformly enriched in 15N and studied by 1H- and 15N-NMR spectroscopy. Relaxation and NOE data allowed determination of the rotational correlation time and indicated more rapid side-chain motion in the native protein and increased segmental motion in the base-denatured protein. The pi nitrogen of the ligand histidine and the indolic nitrogen of the invariant tryptophan both remain protonated and act as proton-donors in hydrogen bonds over a wide pH range and therefore do not contribute to pH-related changes in the midpoint potential. pK values identified by numerous methods in the ferrocytochrome at pH 6.9 and in the ferricytochrome at pH 6.2 arise from His-42. At pH values below the pK, the imidazolium group participates in a salt bridge or in a hydrogen bond with the carboxylate group of the inner propionate of the heme. Loss of the proton causes a local conformational change which alters the midpoint potential. The pK values of the amino terminus and lysines were also determined from pH titrations monitored by 15N-NMR. Similar titrations of the ferricytochrome monitored by 1H-NMR showed structural heterogeneity in that the resonance of heme ring methyl 8 split into a doublet as the pH was raised.