Robert G. Bartsch

[34] Cytochromes: Bacterial

Publisher Summary Absorption spectra measurements are routinely used to assay the cytochromes. Because of the high concentration of strongly absorbing carotenoids and chlorophylls in wild strains of the photosynthetic bacteria, it is difficult to detect cytochrome absorption bands with a hand spectroscope. In addition, the effect of light scattering makes it difficult to measure cytochrome spectra of whole cell suspensions even with many sensitive spectrophotometers. Reduced-minus-oxidized difference spectra between two initially equivalent buffered samples must be resorted to for an indication of the cytochrome content of a suspension of cells or of membrane fragments. With a split-beam spectrophotometer, such as a Cary 14 (or 15) instrument, one of the pair of samples may be left unaltered, chemically oxidized with a small amount of potassium ferricyanide or even sodium hypochlorite, or exposed to photoactive light to test for a light-induced reaction. The other sample may be reduced by endogenous reductants, or by the requisite amount of chemical reductants, such as succinate, ascorbate, NADH, sodium dithionite, or thiol compounds, such as 2-mercaptoethanol or dithiothreitol. Alternatively, a dual wavelength spectrophotometer may be used. The same techniques are applicable to cell-free extracts and purified cytochromes.

Light-induced electron transport in Chromatium strain D II. Light-induced absorbance changes in Chromatium chromatophores

Abstract The light-particle preparations, described previously as derived from chromatophore fractions of Chromatium, exhibit absorbance changes on steady-state illumination with actinic light. Conditions for optimal absorbance changes were established and used to study the effects of variation in redox potential effective at the light-activated electron transport system. The results indicate that at least six components participate in photo-induced changes in oxidation states; two of these can be correlated with the known cytochrome components—cytochrome c−552 and cytochrome c−555. The natures of the remaining components remain to be established. A model is proposed for the electron transport mechanisms activated by light, the salient feature of which is the existence of two functionally different pathways of photo-activated electron movement. One of these involves a pigment-heme protein complex consisting of the 890 mμ active center bacteriochlorophyll coupled to cytochrome c−555, cytochrome cc′ and a third cytochrome (component No. 3); the other consists of a 905 mμ active center bacteriochlorophyll coupled to cytochrome c−552 and a low potential component (P−135). A rationalization of all observations on photometabolism of Chromatium on the basis of this model is presented.

Chemical characterization of high potential iron proteins from Chromatium and Rhodopseudomonas gelatinosa

Chemical and physicochemical studies on the high potential iron proteins from Chromatium and from Rhodopseudomonas gelatinosa reveal a close structural relationship, indicating a new class of non-heme proteins. The 4 residues of cysteine and the 4 moles of iron and labile sulfur, present in both proteins, form part of a chelating complex which absorbs in the visible and in the ultraviolet region. It is suggested that the iron may be attached to the protein by additional linkages involving free carboxyl groups of aspartyl and glutamyl residues, and that this portion of the ligand complex may be mainly responsible for the enhanced ultraviolet absorption. The proteins each consist of a single polypeptide chain with serine and alanine in the N-terminal, and glycine and alanine in the C-terminal position of the Chromatium and Rps. gelatinosa proteins, respectively. The sequence of the first ten residues starting from the N-terminus was established by Edman degradation: Chromatium: H2N-Ser-Ala-Pro-Ala-Asn-Ala-Val-Gly-Ala-Gly… Rps. gelatinosa: H2N-Ala-Pro-Val-Asp-Glu-Lys-Asn-Pro-(Glu)-Ala… The Em (pH 7.0) of +0.35 V for the Chromatium and of +0.33 for the Rps. gelatinosa iron protein clearly distinguishes this group from the “ferredoxins”. The pl′ (0°) was found to be at pH 3.68 and 9.50 for the reduced iron proteins from Chromatium and Rps. gelatinosa, respectively. However, the oxidized form of the Chromatium protein had a pI′ (0°) at pH 3.88. A histidine-iron linkage in the reduced protein is suggested to explain this charge difference of 0.20 pH units as well as the transfer of a single electron observed for this protein. This single histidine was completely dinitrophenylated in the oxidized form but was unavailable in the reduced form of the protein. A convenient approach to chemical molecular weight determination based on end-group and amino acid analyses yielded a value of 10074 for the molecular weight of the Chromatium and 9579 as molecular weight for the Rps. gelatinosa protein. Precoated thin-layer sheets were employed for the rapid separation and identification of phenylthiohydantoin-amino acids on a micro scale using solvents C, D, and E, as introduced for paper chromatography.

The cytochromes of Chlorobium thiosulfatophilum

Three c-type cytochromes (c-551, c-553, c-555) have been isolated and characterized from a strain of the green photosynthetic bacterium Chlorobium thiosulfatophilum. These cytochromes are atypical when compared to horse heart cytochrome c in many properties, among them: oxidation-reduction potential at pH 7.0 (c-551, 135 mV; c-553, 98 mV; c-555, 145 mV), molecular weight (c-551, 45000–60000; c-553, 50000; c-555, 10000) and isolelectric point (c-551, 6.0; c-553, 6.7). No protoheme was detected in whole cells or cell-free extracts.

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2·5 Å resolution

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability

The cytochromes c2 of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c2 functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345-395 mV at pH 5. Closely related cytochromes c2 with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding. The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.

[35] Purification of (4Fe-4S)1—2− ferrodoxins (high-potential iron-sulfur proteins) from bacteria

Publisher Summary This chapter describes the purification of (4Fe-4S) 1-2- ferredoxins (high-potential iron–sulfur proteins) from bacteria. A soluble, high-potential, iron–sulfur protein (HiPIP) was first recognized in extracts of the photosynthetic bacterium Chrornatium vinosum and subsequently in only a few other photosynthetic bacterial species out of many examined: Rhodopseudomonas gelatinosa , Thiocapsa pfennigii , Rhodospirillum tenue , a marine purple sulfur isolate designated HOL-1, in very small amounts Rhodomicrobium vannielii , and in one nonphotosynthetic bacterium, a denitrifying, halophilic Paracoccus specie. Because Fe–S proteins are now classified on the basis of the chemical structure of their chromophoric group, the bacterial HiPIPs are included as a subgroup of the (4Fe-4S) ferredoxins. HiPIP is best stored in the reduced state. Oxidized HiPIP becomes autoreduced, presumably at the expense of reductant provided by disintegrating 4Fe-4S clusters. Although initial concentrations of HiPIPs in crude extracts are accurately determined, it is estimated that no more than 50% of the proteins is lost during the purification.

pH dependence of the oxidation-reduction potential of cytochrome c2.

The pH dependence of the spectra and of the oxidation-reduction potential of three cytochromes c2, from Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodomicrobium vannielii, were studied. A single alkaline pK was observed for the spectral changes in all three ferricytochromes. In Rps. capsulata cytochrome c2 this spectroscopic pK corresponds to the pK observed in the dependence of oxidation-reduction potential on pH. For the other two cytochromes the oxidation-reduction potential showed a complex dependency on pH which can be fitted to theoretical curves involving three ionizations. The third ionization corresponds to the ionization observed in the spectroscopic studies but the first two occur without changes in the visible spectra. The possible structural bases for these ionizations are discussed.

ATYPICAL SOLUBLE HAEM PROTEINS FROM A STRAIN OF RHODOPSEUDOMONAS PALUSTRIS SP.

Abstract A strain of Rhodopseudomonas has been found to contain atypical specimens of the Rhodopseudomonas haem-type protein and c -type cytochrome. These proteins exhibit anomalies with respect to solubility, spectroscopic behavior, midpoint potential, as well as the presence of blocked NH 2 -terminal groups. The main findings are the wholly uncharacteristic high midpoint potential (about 105 mV) and a relatively low degree of auto-oxidizability of the RHP-type protein.

The distribution of soluble metallo-redox proteins in purple phototrophic bacteria

A comparison is made of types and distribution of cytochromes and certain ferredoxins (HiPIP) among photosynthetic bacteria. These are subdivided as to the type of reaction center each species is believed to contain. The proteins listed are assumed to be of periplasmic origin. Interrelationships suggested by the comparison are discussed.