Spectroscopic characterization of nitrosylheme in nitric oxide complexes of ferric and ferrous cytochrome c' from photosynthetic bacteria.

Reactions of ferric and ferrous cytochromes c' from four photosynthetic bacteria (Rhodobacter capsulatus ATCC 11166, Rhodopseudomonas palustris ATCC 17001, Rhodospirillum rubrum ATCC 11170, and Chromatium vinosum ATCC 17899) with nitric oxide have been investigated by electronic absorption and electron paramagnetic resonance spectroscopies. The heme iron(III) of these ferric cytochromes c' has been recently reported to be in a quantum mechanically admixed (S = 5/2, 3/2) state [Fujii, S., Yoshimura, T., Kamada, H., Yamaguchi, K., Suzuki, S., Shidara, S. and Takakuwa, S. (1995) Biochim. Biophys. Acta 1251, 161-169]. The affinity of ferric cytochromes c' for NO among these bacterial species (C. vinosum > Rps. palustris approximately Rb. capsulatus >> R. rubrum) was apparently related to the S = 3/2 content in the or der. In the reaction of ferrous cytochrome c' with NO, six- and five-coordinated nitrosylhemes, which represent species with and without a ligand at the axial position trans to nitrosyl group, have been formed. The content of six-coordinated nitrosylheme in NO-ferrous cytochrome c' has been determined to be Rb. capsulatus approximately Rps. palustris > C. vinosum < R rubrum, suggesting that a stability of iron-to-histidine bond decreases with this order. The NO reactions of ferric and ferrous cytochromes c' from photosynthetic bacteria have been compared with those of cytochromes c' from denitrifying bacteria.

Excitation delocalization over the whole core antenna of photosynthetic purple bacteria evidenced by non-linear pump-probe spectroscopy.

Anomalously high values of photoinduced absorption changes were revealed in the antenna of photosynthetic purple bacteria. They were found to be 4-16 times greater at the bleaching peak of the antenna than at the bleaching peak of the BChl dimer of the reaction center. This is direct proof of excitation delocalization over many pigment molecules. Calculations according to the model of exciton delocalization over all core antenna BChls allow one to explain the observed phenomenon.

Effect of hydration on the structure, dynamics and function of photosynthetic membranes of purple bacteria.

NMR spectra and relaxation times T1 and T2 for 31P in membranes of Rhodobacter sphaeroides were investigated at different relative humidity levels. The results are compared to the hydration curves, fatty acid composition and the structure-dynamic and functional characteristics of the membranes of photosynthetic bacteria Rb. sphaeroides, Rhodospirillum rubrum and Ectothiorhodospira shaposhnikovii. The differences in the state of lipid phase of these membranes are revealed under low humidity, and this is conducive to variability of their structural dynamic and functional characteristics during the hydration process. Based on the results obtained and the data on model systems, four stages of hydration process are distinguished with different effects on the structure and dynamics of membrane components. These stages are: hydration of a portion of polar groups, involvement of water molecules in the hydrogen bonds within macromolecules and the lipid phase, hydration of all polar groups with the appearance of water with high dielectric constant thus making possible the lateral diffusion within the membrane and realization, through water participation, of conditions within organelles and cells required for the process regulation at these levels. The mechanism of water action on various membrane components and their dynamics at each stage are discussed, as well as the effect of different types of motion on the efficiency and regulation of electron transport in the photosynthetic chain of the membranes studied.

Methods of isolation of reaction center preparations from photosynthetic purple bacteria.

Various modern methods of isolation of functionally active membrane complexes of the reaction centers (RC) from photosynthetic purple bacteria are reviewed. Special attention is given to the methods of RC isolation from bacteria which are widely used in experimental practice. The analysis includes the main steps of RC isolation, evaluation of purity of the resultant preparation, and characterization of its functional activity. Besides description of conventional methods of RC isolation based on ion-exchange chromatography and hydroxyapatite chromatography, some other methods such as affinity chromatography and high-performance chromatography at high and fine pressure are also considered.

Structural and spectral characterisation of the antenna complexes of Rhodocyclus gelatinosus. Indications of a hairpin-like-arranged antenna apoprotein with an unusually high alanine content.

The core antenna/reaction-centre complex RC-B875 and the peripheral antenna complex B800-850 of the two strains DSM 149 and DSM 151 of the purple non-sulphur bacterium Rhodocyclus gelatinosus have been isolated from photosynthetic membranes by means of lauryl-N,N-dimethyl-amineoxide as a detergent and subsequent sucrose-gradient centrifugation. The two complexes were characterised spectroscopically by absorption and circular dichroism (CD) spectroscopy at room temperature. CD measurements revealed very weak signals for the core antenna B875 whereas for the peripheral antenna B800-850, a strong biphasic CD signal was observed, attributable to the B850 pigments. There is apparently no CD signal present for the B800 pigments. The core and the peripheral antenna complex are built up by a distinct alpha/beta-polypeptide pair. The pigment/protein ratio in the peripheral antenna complex is 3 bacteriochlorophyll/(alpha/beta)-polypeptide pair. The amino acid sequences of the alpha and beta polypeptides of both complexes from the two strains of Rc. gelatinosus were established by automated Edman degradation, chemical and enzymic digestion, amino acid composition analyses and carboxypeptidase digestion. In the case of the beta polypeptides, the amino acid sequence determination was confirmed by ion-spray MS of the isolated antenna apoproteins. The inter-strain (DSM 149 and 151) positional identity between the equivalent apoproteins is extremely large and varies in the range 90-100%. The B875-beta polypeptide from Rc. gelatinosus exhibits shortened C-termini, as detected for the analogous antenna apoproteins of Rhodobacter sphaeroides and Rhodobacter capsulatus, which can be correlated with weak core antenna near-infrared CD signals. However, the B800-850-alpha polypeptide of Rc. gelatinosus, with 71 amino acids, exhibits an extended C-terminal portion indicative of the formation of a second transmembrane domain, which so far has not been observed for bacterial antenna apoproteins. This part of the molecule is extremely rich in alanine and proline residues. All the sequenced antenna apoproteins of Rc. gelatinosus exhibit a characteristic membrane-buried histidine which is thought to ligate the B875 or the B850 pigments. In the B800-850-beta apoprotein, a second, so far beta-antenna-apoprotein-specific histidine, is replaced by a glutamine residue. A careful inspection of the determined antenna structures of Rc. gelatinosus revealed some remarkable structural similarities within presumed cofactor-binding sites of Fe-S-type-reaction-centre apoproteins, indicating possible basic structural motifs for complexing bacteriochlorophyll molecules.

Sequence evidence for strong conservation of the photoactive yellow proteins from the halophilic phototrophic bacteria Chromatium salexigens and Rhodospirillum salexigens.

The photoactive yellow proteins (PYP) have been found to date only in three species of halophilic purple phototrophic bacteria. They have photochemical activity remarkably similar to that of the bacteria rhodopsins. In contrast to rhodopsins, however, the PYPs are small water-soluble proteins. We now report the complete amino acid sequences of Rhodospirillum salexigens and Chromatium salexigens PYP which allow comparison with the known sequence and three-dimensional structure of the prototypic protein from Ectothiorhodospira halophila. Although isolated from three different families of bacteria, the PYP sequences are 70-76% identical. All three contain 125 amino acid residues, and no insertions or deletions are necessary for alignment. This is a remarkable result when it is considered that electron transfer proteins from these purple bacterial species are only 25-40% identical and that insertions and deletions are needed for their proper alignment. It thus appears that PYP has the same important function in each of the purple bacteria and that most of the amino acid residues are necessary to maintain structure and function. By most standards, PYP would be called a "slowly evolving protein". R. salexigens PYP is uniquely degraded by proteolysis at low ionic strength, probably as a consequence of unfolding due to electrostatic repulsion of the excess negative charge. Therefore it may also be classified as a "halophilic protein".

The antenna complexes of the purple non-sulfur photosynthetic bacterium Rhodocyclus tenuis. Structural and spectral characterization.

The photoreceptor complex (B885-RC) and the peripheral antenna complex (B800-860) were isolated from photosynthetic membranes of the purple non-sulfur bacterium Rhodocyclus tenuis DSM 109 using a detergent combination of Deriphate-160 and octyl glucoside and subsequent linear sucrose gradient centrifugation. The two complexes were characterized by room-temperature absorption, circular dichroism and fluorescence spectroscopy. The B800-860 complex has a more red-shifted B860 absorbance band. The alpha,beta-polypeptides were purified with a reverse-phase HPLC system and resolved at a ratio of 1:1 in the B800-860 complex and at an overall ratio of 1:1 for the B885-RC complex. The complete amino acid sequences of the alpha and beta polypeptides of the B800-860 and B885-RC complexes were determined by micro-sequencing analysis and mass spectrometry. The B800-860-alpha polypeptide possesses an identical N-terminal domain (the first 15 residues) to Rhodobacter sphaeroides B800-850-alpha polypeptide. The central hydrophobic and C-terminal domains of the B800-860-alpha,beta polypeptides show a number of B870/880-like structural elements in which, of special interest, is the WWSEF cluster in the C-terminal domain of the B800-860-alpha polypeptide which is very similar to the WWEF cluster in the same region of Rhodopseudomonas viridis B1015-alpha polypeptide. The more red-shifted absorption characteristic of the 860-nm bacteriochlorophylls could most probably be related to the B870/880-like polypeptide features in the central hydrophobic domains and the C-terminal domains of the B800-860-alpha,beta polypeptides. The hydropathy plot of the B800-860-alpha polypeptide exhibits an extended C-terminal hydrophobic segment indicative of a second membrane-contacting domain, which has not been found in the antenna polypeptides of the purple bacteria with intracytoplasmic membranes. Further sequence analysis revealed the existence of multiple forms of the B885-alpha,beta polypeptides, the B885-alpha 1,alpha 2 polypeptides, and the B885-beta 1,beta 2 polypeptides. The B885-Alpha 2 polypeptide shows an identical sequence to the B885-alpha 1 polypeptide, but it is 12 amino acid residues shorter than the B885-alpha 1 polypeptide at the C-terminal. The two species of the B885-beta polypeptides were identified as an identical sequence with only one amino acid residue variation at sequence position 34, where the B885-beta 1 has a valine residue and the B885-beta 2 polypeptide an isoleucine residue. The possible correlation between the intensity of the near-infrared circular dichroic signal and the specific structural features of the alpha and beta core antenna polypeptides is also discussed.

Characterization of an improved reaction center preparation from the photosynthetic green sulfur bacterium Chlorobium containing the FeS centers FA and FB and a bound cytochrome subunit.

A photosynthetic reaction center complex was prepared from the green sulfur bacterium Chlorobium by solubilization of chlorosome-depleted membranes with lauryl maltoside, followed by anion-exchange chromatography and molecular sieve chromatography. The purified complex was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, optical spectroscopy, and EPR spectroscopy. The major bands migrated at apparent molecular masses of 50, 42, and 32 kDa (heme-staining) and additional weaker bands at 22, 15, and 12 kDa. The isolated reaction center complex contained about 40 bacteriochlorophyll alpha molecules per primary electron donor, P840, assayed by photooxidation. It was competent in stable low-temperature photoreduction of the FeS centers FA and FB. The spectra of these acceptors and their low-temperature photochemistry in the purified complex were the same as found in intact Chlorobium membranes and similar to what had been described for photosystem I from plants. Membrane-bound cytochrome c553 copurified with the reaction center complex. A ratio of about four hemes per P840 was determined. This result indicates that cytochrome c553 that is closely associated with the reaction center is a tetraheme cytochrome, as described for some purple bacteria.

Expression and characterization of recombinant Rhodocyclus tenuis high potential iron-sulfur protein.

The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein were similar to those of the native protein. EPR of the oxidized protein shows g values of 2. 11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a [Fe(4)S(4)] cluster in the holoprotein. The observed mass of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein. The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating correct folding and incorporation of the iron-sulfur cluster.