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R G Bartsch

Publications and source records attributed to R G Bartsch.

At least 37 records · Page 2Linked to original sources
PubMed1991

Redox potentials of flavocytochromes c from the phototrophic bacteria, Chromatium vinosum and Chlorobium thiosulfatophilum.

The redox potentials of flavocytochromes c (FC) from Chromatium vinosum and Chlorobium thiosulfatophilum have been studied as a function of pH. Chlorobium FC has a single heme which has a redox potential of +98 mV at pH 7 (N = 1) that is independent of pH between 6 and 8. The average two-electron redox potential of the flavin extrapolated to pH 7 is +28 mV and decreases 35 mV/pH between pH 6 and 7. The anionic form of the flavin semiquinone is stabilized above pH 6. The redox potential of Chromatium FC is markedly lower than for Chlorobium. The two hemes in Chromatium FC appear to have a redox potential of 15 mV at pH 7 (N = 1), although they reside in very different structural environments. The hemes of Chromatium FC have a pH-dependent redox potential, which can be fit in the simplest case by a single ionization with pK = 7.05. The flavin in Chromatium FC has an average two-electron redox potential of -26 mV at pH 7 and decreases 30 mV/pH between pH 6 and 8. As with Chlorobium, the anionic form of the flavin semiquinone of Chromatium FC is stabilized above pH 6. The unusually high redox potential of the flavin, a stabilized anion radical, and sulfite binding to the flavin in both Chlorobium and Chromatium FCs are characteristics shared by the flavoprotein oxidases. By analogy with glycolate oxidase and lactate dehydrogenase for which there are three-dimensional structures, the properties of the FCs are likely to be due to a positively charged amino acid side chain in the vicinity of the N1 nitrogen of the flavin.

Bacteria↗
PubMed1991

Amino acid sequences of Euglena viridis ferredoxin and cytochromes c.

The Order Euglenida comprises many species and perhaps 40 genera, but almost all biochemical and genetic studies have been limited to a single species. Euglena gracilis, because of its ease of growth in the laboratory. Sequence studies of chloroplast and mitochondrial proteins from E. gracilis show that they have diverged widely from other eukaryotic lines. In the present paper we report the sequences of three proteins from another euglenoid, Euglena viridis, using material isolated from a natural bloom. The mitochondrial cytochrome c shows more than 90% sequence identity with that from E. gracilis, and contains the same characteristic features. The chloroplast cytochrome c6 has diverged to a greater extent and shows only 77% identity. The chloroplast ferredoxin from E. viridis is similar in sequence to those of cyanobacteria and algal chloroplasts, with sequence identities of up to 75%. Details of the purification, analysis and sequence determination experiments on the peptides have been deposited as Supplementary Publication SUP 50163 (32 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Amino Acid Sequence↗
PubMed1991

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.

Cytochrome c Group↗
PubMed1990

Proton NMR study of the comparative electronic/magnetic properties and dynamics of the acid in equilibrium with alkaline transition in a series of ferricytochromes c'.

The proton NMR spectra of ferricytochrome c' from Rhodopseudomonas palustris, Rhodospirillum molischianum, Rhodospirillum rubrum, and Chromatium vinosum have been investigated for the purpose of further elucidating the common spectral and/or structural properties for this subclass of cytochromes in the acidic and alkaline forms, and to characterize in detail the dynamics and structural basis for this acid in equilibrium with alkaline transition. The identification of strongly upfield-shifted meso-H peaks in all but C. vinosum ferricytochrome c' at weakly acidic to neutral pH is consistent with, but not proof for, S = 3/2 character for the spin state of C. vinosum, but argues for primarily S = 5/2 character for the other three proteins. Hence, we conclude that the quantum mechanically mixed S = 3/2, S = 5/2 spin ground state of neutral pH C. vinosum ferricytochrome c' is an anomaly rather than a characteristic of this class of proteins. The 1H NMR spectra of ferricytochromes c' at alkaline pH again exhibit strong similarities among all members except that for C. vinosum. Two pK values are observed for ferricytochrome c' for R. molischianum and C. vinosum, of which the higher value pK is accompanied by significant line broadening, as found earlier for the proteins from both R. rubrum and R. palustris. Detailed analysis of the exchange line broadening for all four proteins reveals that hydrolysis is the rate-limiting step, with base catalysis occurring at about the same rate in the diffusion control limit for all four proteins. The variable first order dissociation rates of the alkaline species reveal differential stabilities of that species in the order R. palustris greater than R. molischianum greater than R. rubrum much greater than C. vinosum. The rates of exchange of the axial His imidazole labile proton was determined by linewidth and saturation transfer analysis and shown to occur via base catalysis at the same diffusion control rate as found for the acid----alkaline transition for the oxidized protein, and support the proposal that the acid----alkaline transition involves simply the abstraction of a proton from the neutral His imidazole to yield an imidazolate.

Chromatium↗
PubMed1990

Complete amino acid sequence of the cytochrome subunit and amino-terminal sequence of the flavin subunit of flavocytochrome c (sulfide dehydrogenase) from Chlorobium thiosulfatophilum.

The complete amino acid sequence of the 86-residue heme subunit of flavocytochrome c (sulfide dehydrogenase) from the green phototrophic bacterium Chlorobium thiosulfatophilum strain Tassajara has been determined as follows: APEQSKSIPRGEILSLSCAGCHGTDGKSESIIPTIYGRSAEYIESALLDFKSGA- RPSTVMGRHAKGYSDEEIHQIAEYFGSLSTMNN. The subunit has a single heme-binding site near the N terminus, consisting of a pair of cysteine residues at positions 18 and 21. The out-of-plane ligands are apparently contributed by histidine 22 and methionine 60. The molecular weight including heme is 10,014. The heme subunit is apparently homologous to small cytochromes c by virtue of the location of the heme-binding site and its extraplanar ligands. However, the amino acid sequence is closer to Paracoccus sp. cytochrome c554(548) (37%) than it is to the heme subunit from Pseudomonas putida p-cresol methylhydroxylase flavocytochrome c (20%). The flavocytochrome c heme subunit is only 14% similar to the small cytochrome c555 also found in Chlorobium. Secondary structure predictions suggest N- and C-terminal helices as expected, but the midsection of the protein probably folds somewhat differently from the small cytochromes of known three-dimensional structure such as Pseudomonas cytochrome c551. Analyses of the residues near the exposed heme edges of the cytochrome subunits of P. putida and C. thiosulfatophilum flavocytochromes c (assuming homology to proteins of known structure) indicate that charged residues are not conserved, suggesting that electrostatic interactions are not involved in the association of the heme and flavin subunits. The N-terminal sequence of the flavoprotein subunit of flavocytochrome has also been determined. It shows no similarity to the comparable region of the p-cresol methylhydroxylase flavoprotein subunit from P. putida. The flavin-binding hexapeptide, isolated and sequenced earlier (Kenney, W. C., McIntire, W., and Yamanaka, T. (1977) Biochim. Biophys. Acta 483, 467-474), is situated at positions 40-46.

Amino Acid Sequence↗
PubMed1990

Soluble cytochromes and ferredoxins from the marine purple phototrophic bacterium, Rhodopseudomonas marina.

Four soluble c-type cytochromes, the high redox potential 4-Fe-S ferredoxin known as HiPIP, a large molecular weight 2-Fe-S ferredoxin and a 4-Fe-S 'bacterial' ferredoxin, were isolated from extracts of two strains of Rps. marina. Cytochrome c-550, cytochrome c' and cytochrome c-549 were previously described, and we have extended their characterization. Cytochrome c-558, which has not previously been observed in Rps. marina, appears to be a low-spin isozyme of the more commonly observed high-spin cytochrome c'. HiPIP, which was not observed in previous work, was found to be abundant in Rps. marina. The 2-Fe-S ferredoxin, which has previously been observed only in Rps. palustris, has a native size greater than 100 kDa and a subunit size of 17 kDa. The 'bacterial' ferredoxin appears to have only a single four-iron-sulfur cluster. We examined photosynthetic membranes by difference spectroscopy and found abundant c-type cytochromes. Approximately one-quarter of the heme can be reduced by ascorbate and the remainder by dithionite. There is 2 nm difference between the high-potential heme (554 nm) and the low (552 nm). These characteristics resemble those of the tetraheme reaction center cytochrome of Rps. viridis. In addition to the electron transfer components, we found small amounts of a fluorescent yellow protein which has spectral resemblance to a photoactive yellow protein from Ec. halophila.

Bacterial Proteins↗
PubMed1990

Soluble cytochromes and a photoactive yellow protein isolated from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salexigens.

Three soluble cytochromes were found in two strains of the halophilic non-sulfur purple bacterium Rhodospirillum salexigens. These are cytochromes C2, C and c-551. Cytochrome C2 was recognized by the presence of positive charge at the site of electron transfer (measured by laser flash photolysis), although the protein has an overall negative charge (pI = 4.7). Cytochrome C2 has a high redox potential (300 mV) and is monomeric (13 kDa). Cytochrome c was recognized from its characteristic absorption spectrum. It has a redox potential of 95 mV, an isoelectric point of 4.3, and is isolated as a dimer (33 kDa) of identical subunits (14 kDa), a property which is typical of this family of proteins. R. salexigens cytochrome c-551 has an absorption spectrum similar to the low redox potential Rb. sphaeroides cytochrome c-551.5. It also has a low redox potential (-170 mV), is very acidic (pI = 4.5), and is monomeric (9 kDa), apparently containing 1 heme per protein. The existence of abundant membrane-bound cytochromes c-558 and c-551 which are approximately half reduced by ascorbate and completely reduced by dithionite suggests the presence of a tetraheme reaction center cytochrome in R. salexigens, although reaction centers purified in a previous study (Wacker et al., Biochim. Biophys. Acta (1988) 933, 299-305) did not contain a cytochrome. The most interesting observation is that R. salexigens contains a photoactive yellow protein (PYP), previously observed only in the extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila. The R. salexigens PYP appears to be slightly larger than that of Ec. halophila (16 kDa vs. 14 kDa). Otherwise, these two yellow proteins have similar absorption spectra, chromatographic properties and kinetics of photobleaching and recovery.

Ascorbic Acid↗
PubMed1989

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554.

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.

Aerobiosis↗
PubMed1987

Kinetics of reduction by free flavin semiquinones of algal cytochromes and plastocyanin.

It had been shown that plastocyanin and cytochrome c-553 are functionally interchangeable in algae and that the physiological electron transfer reactions are sensitive to ionic strength. The isoelectric points of these proteins range from very acidic to basic depending upon species, and naturally occurring amino acid substitutions of charged residues have been shown to affect the kinetics of electron transfer, presumably through alteration of protein net charge. We have now shown that these naturally occurring amino acid substitutions also affect the kinetics of nonphysiological electron transfer reactions, and that we can quantitate the extent of nonconservation of charge. The reduction of plant and algal proteins by FMN semiquinone is sensitive to ionic strength and the effects can be correlated with net protein charge with regard to sign, but not to magnitude, with the charge at the site of electron transfer varying from +3 through 0 to -3. We had previously observed in a large variety of electron transfer proteins from bacteria (G. Tollin, T. E. Meyer, and M. A. Cusanovich (1986) Biochim. Biophys. Acta 853, 29-41) that charge localized at the site of electron transfer, rather than net protein charge, was more likely to affect kinetics. This also appears to be the case with the algal proteins. By comparison of protein structures, we have been able to predict which substitutions are likely to be responsible for the kinetic effects in the algal proteins and to discuss the implications of such changes for function.

Amino Acid Sequence↗
PubMed1986

Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin.

Rate constants have been measured for the reactions of a series of high-spin cytochromes c' and their low-spin homologues (cytochromes c-554 and c-556) with the semiquinones of free flavins and flavodoxin. These cytochromes are approximately 3 times more reactive with lumiflavin and riboflavin semiquinones than are the c-type cytochromes that are homologous to mitochondrial cytochrome c. We attribute this to the greater solvent exposure of the heme in the c'-type cytochromes. In marked contrast, the cytochromes c' are 3 orders of magnitude less reactive with flavodoxin semiquinone than are the c-type cytochromes. We interpret this result to be a consequence of the location of the exposed heme in cytochrome c' at the bottom of a deep groove in the surface of the protein, which is approximately 10-15 A deep and equally as wide. While free flavins are small enough to enter the groove, the flavin mononucleotide (FMN) prosthetic group of flavodoxin is apparently prevented by steric constraints from approaching the heme more closely than approximately 10 A without dynamic structural rearrangements. Most cytochromes c' are dimeric, but a few are monomeric. The three-dimensional structure of the Rhodospirillum molischianum cytochrome c' dimer suggests that the heme should be more exposed in the monomer than in the dimer, but no relationship is observed between intrinsic reactivity toward free flavin semiquinones and the aggregation state of the protein. Likewise, there is no evidence that the spin state or ligand field of the iron has any effect on intrinsic reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1986

The amino acid sequence of cytochrome c-555 from the methane-oxidizing bacterium Methylococcus capsulatus.

The amino acid sequence of the cytochrome c-555 from the obligate methanotroph Methylococcus capsulatus strain Bath (N.C.I.B. 11132) was determined. It is a single polypeptide chain of 96 residues, binding a haem group through the cysteine residues at positions 19 and 22, and the only methionine residue is a position 59. The sequence does not closely resemble that of any other cytochrome c that has yet been characterized. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50131 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment.

Amino Acid Sequence↗
PubMed1985

The major soluble cytochromes of the obligately aerobic sulfur bacterium, Thiobacillus neapolitanus.

Four cytochromes were isolated from soluble extracts of the aerobic sulfur bacterium, Thiobacillus neapolitanus. The two most abundant proteins were purified to homogeneity and thoroughly characterized. Cytochrome c-554 (547) is a monomeric, small molecular weight protein which is unusual in having two well-resolved alpha peaks in UV-visible absorption spectra. The redox potential is 208 mV. Native cytochrome c-549 is oligomeric, but has a subunit size of about 26,000. The yield of this protein could be improved dramatically by washing membranes with 30% ammonium sulfate, but the material solubilized by this method had a larger native molecular weight than that in the initial 0.1 M Tris-Cl extract and behaved differently on chromatography. The properties of cytochrome c-549 including subunit size and UV-visible absorption spectra are similar to mitochondrial cytochrome c1 and chloroplast cytochrome f, which suggests that it may be a modified form of the predominant membrane cytochrome. Based on cytochrome content, it is suggested that T. neapolitanus is not closely related to other thiobacilli.

Amino Acids↗
PubMed1983

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins.

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.

Adrenodoxin↗
PubMed1983

Proton nuclear magnetic resonance studies of the ligation states of the monomeric ferricytochrome c' from Rhodopseudomonas palustris. Modulation of axial histidine bonding via variable proton donation.

The monomeric ferricytochrome c' from Rhodopseudomonas palustris strain 37 has been examined by 1H NMR spectroscopy at 45 degrees C and 360 MHz and at 55 degrees C and 200 MHz in 2H2O. The pH-dependent characteristics of the spectra have been analyzed in terms of two pK values at approximately 6 and approximately 8, the latter of which appears to correspond to the previously observed transition between an acidic species and a neutral species for this class of proteins. Previously determined rate constants (J. T. Jackson, G. N. La Mar, R. G. Bartsch, and M. A. Cusanovich, manuscript in preparation) permit, for the first time, connection between the downfield hyperfine shifted resonances of the two forms by spectral simulation. Contrary to previous suggestions (Emptage, M. H., Xavier, A. V., Wood, J.M., Alsaadi, B. M., Moore, G. R., Pitt, R. C., Williams, R. J. P., Ambler, R. P., and Bartsch, R. G. (1981) Biochemistry 20, 58-64; La Mar, G. N., Jackson, J. T., and Bartsch, R., (1981) J. Am. Chem. Soc. 103, 4405-4410), the data indicate that no additional ligand is involved in the transition but that the bonding between the iron and the axial histidyl ligand is severely altered. It is shown that this is consistent with extensive imidazolate character for the axial ligand in the neutral form.

Cytochrome c Group↗
PubMed1982

Metal coordination centres of class II cytochromes c.

The class II cytochromes Rhodospirillum molischianum cytochrome c', Rhodopseudomonas palustris cytochrome C556 and Agrobacterium tumefaciens (B2a) cytochrome c556 have been investigated with a variety of spectroscopic techniques. The cytochrome c' was found to be high-spin and the two cytochromes c556 were found to be mainly low-spin and sx-coordinate with the fifth and sixth ligands being histidine and methionine. The implications of the different types of iron coordination are discussed.

Cytochrome c Group↗
PubMed1981

Nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome c'.

Cytochrome c' from Rhodospirillum rubrum has been studied by proton magnetic resonance (NMR) at 270 MHz. The pH and temperature-dependence properties as well as proton water relaxation enhancement and bulk susceptibility measurements were examined. We conclude that the fifth ligand to the iron is histidine. The pH-dependent shift of the heme methyl resonances of the ferric protein shows pKa's at 5.8 and 8.7. The low-pH equilibrium causes only minor changes in the properties of the protein. However, the high-pH equilibrium causes large changes throughout the NMR spectra which correlate with the reported visible spectral changes. These NMR spectral changes are compared with the low-temperature EPR and Mössbauer spectroscopic data. Analyses of the NMR data show that a second histidine, which is present in the sequence of c' from R. rubrum but is not conserved in other cytochromes c', is not a "distal" histidine. The nature of the sixth ligand and the significance of the high-pH transition are discussed.

Cytochrome c Group↗
PubMed1981

Amino acid sequences of bacterial cytochromes c' and c-556.

The cytochrome c' are electron transport proteins widely distributed in photosynthetic and aerobic bacteria. We report the amino acid sequences of the proteins from 12 different bacterial species, and we show by sequences that the cytochromes c-556 from 2 different bacteria are structurally related to the cytochromes c'. Unlike the mitochondrial cytochromes c, the heme binding site in the cytochromes c' and c-556 is near the COOH terminus. The cytochromes c-556 probably have a methionine sixth heme ligand located near the NH2 terminus, whereas the cytochromes c' may be pentacoordinate. Quantitative comparison of cytochrome c' and c-556 sequences indicates a relatively low 28% average identity.

Amino Acid Sequence↗