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R C Prince

Publications and source records attributed to R C Prince.

At least 37 records · Page 2Linked to original sources
PubMed1996

The membrane-bound cytochrome cy of Rhodobacter capsulatus can serve as an electron donor to the photosynthetic reaction of Rhodobacter sphaeroides.

Rhodobacter capsulatus has two different pathways for reduction of the photo-oxidized reaction center, one using water-soluble cytochrome c2, the other via membrane-associated cytochrome cy. Rhodobacter sphaeroides differs in that it lacks a cytochrome cy homologue capable of functioning in photosynthetic electron transfer; cytochrome c2 is thus the sole electron carrier, and is required for photosynthetic (Ps+) growth. Genetic evidence indicates that cytochrome cy of R. capsulatus can complement a Ps- cytochrome-c2-deficient mutant of R sphaeroides (Jenny, F.E. and Daldal, F (1993). EMBO J. 12, 1283-1292). Here, we show that it transfers electrons from cytochrome bc1 complex to the reaction center in R. sphaeroides, albeit at a lower rate than that catalyzed by the endogenous cytochrome c2. When cytochrome cy is expressed in R. sphaeroides in the presence of cytochrome c2, there is an increase in the amount of photo-oxidizable c-type cytochrome. In the absence of cytochrome c2, electron transfer via cytochrome cy shows significantly different kinetics for reaction center reduction and cytochrome c oxidation. These findings further establish that cytochrome cy, the electron carrier permitting soluble cytochrome c2-independent photosynthetic growth in R. capsulatus, can function in a similar capacity in R. sphaeroides.

Cytochrome c Group↗
PubMed1995

Alteration of axial coordination by protein engineering in myoglobin. Bisimidazole ligation in the His64-->Val/Val68-->His double mutant.

Pig and human myoglobin have been engineered to reverse the positions of the distal histidine and valine (i.e. His64(E7)-->Val and Val68(E11)-->His). Spectroscopic and ligand binding properties have been measured for human and pig H64V/V68H myoglobin, and the structure of the pig H64V/V68H double mutant has been determined to 2.07-A resolution by x-ray crystallography. The crystal structure shows that the N epsilon of His68 is located 2.3 A away from the heme iron, resulting in the formation of a hexacoordinate species. The imidazole plane of His68 is tilted relative to the heme normal; moreover it is not parallel to that of His93, in agreement with our previous proposal (Qin, J., La Mar, G. N., Dou, Y., Admiraal, S. J., and Ikeda-Saito, M. (1994) J. Biol. Chem. 269, 1083-1090). At cryogenic temperatures, the heme iron is in a low spin state, which exhibits a highly anisotropic EPR spectrum (g1 = 3.34, g2 = 2.0, and g3 < 1), quite different from that of the imidazole complex of metmyoglobin. The mean iron-nitrogen distance is 2.01 A for the low spin ferric state as determined by x-ray spectroscopy. The ferrous form of H64V/V68H myoglobin shows an optical spectrum that is similar to that of b-type cytochromes and consistent with the hexacoordinate bisimidazole hemin structure determined by the x-ray crystallography. The double mutation lowers the ferric/ferrous couple midpoint potential from +54 mV of the wild-type protein to -128 mV. Ferrous H64V/V68H myoglobin binds CO and NO to form stable complexes, but its reaction with O2 results in a rapid autooxidation to the ferric species. All of these results demonstrate that the three-dimensional positions of His64 and Val68 in the wild-type myoglobin are as important as the chemical nature of the side chains in facilitating reversible O2 binding and inhibiting autooxidation.

Animals↗
PubMed1995

Mutation of the Ser2 codon of the light-harvesting B870 alpha polypeptide of Rhodobacter capsulatus partially suppresses the pufX phenotype.

The exact function of the pufX gene product of Rhodobacter capsulatus is uncertain, but deletion of the pufX gene renders cells incapable of phototrophic growth on a minimal medium, and photosynthetic electron transfer is impaired in vitro. However, suppressor mutants that are able to grow phototropically are readily isolated. Two such suppressor mutants were characterized as to their phototrophic growth properties, their fluorescence at different incident light intensities, the integrity of their chromatophores, and their abilities to generate a transmembrane potential. We found that the photosynthetic apparatus in the suppressor mutants was less stable than that of the pseudo-wild-type and primary mutant strains and that the suppressor mutants used light energy less efficiently than the pseudo-wild-type strain. Therefore, the suppressor strains are more precisely designated partial suppressor mutants. The locations and sequences of the suppressor mutations were determined, and both were found to change the second codon of the pufA gene. It is hypothesized that the serine residue specified by this codon is important in interactions between the B870 alpha protein and other membrane-bound polypeptides and that suppressor mutations at this position partially compensate for loss of the PufX protein. A model is proposed for the function of the PufX protein.

Amino Acid Sequence↗
PubMed1994

Roles of the soluble cytochrome c2 and membrane-associated cytochrome cy of Rhodobacter capsulatus in photosynthetic electron transfer.

Genetic evidence indicates that Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center (RC) [Jenney, F. E., & Daldal, F. (1993) EMBO J. 12, 1283-1292]. One pathway is via the water soluble cytochrome (cyt) c2, and the other is via a novel, membrane-associated c-type cytochrome, cyt cy, now believed to be identical to the cyt cx of Jones et al. [Jones, M. R., et al. (1990) Biochim. Biophys. Acta 975, 59-66] and c354 of Zannoni et al. [Zannoni, D., et al. (1992) Arch. Microbiol. 157, 367-374]. Mutants lacking either cyt c2, cyt cy, or the bc1 complex, as well as various combinations, were utilized to probe the functional role of these cytochromes in electron transfer. Data obtained by monitoring flas induced electron transfer kinetics in the RC, cyt c pool, cyt b, and the carotenoid band shift indicate that there are two pathways for electron transfer from the bc1 complex to the RC in R. capsulatus, one via cyt c2 and the other through cty cy. The two pathways show strikingly different kinetics for RC reduction and cyt c oxidation, and both are present in the wild-type strain MT-1131. After genetic inactivation of both cyt c2 and cyt cy there remains no flash oxidizible c-type cytochrome, and inactivation of cyt cy rather than cyt c2 has a more pronounced effect on the extent of the light-induced membrane potential under the conditions tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1993

X-ray absorption spectroscopy of oriented cytochrome oxidase.

The polarized X-ray absorption spectra of the copper, iron and zinc sites of mitochondrial cytochrome oxidase in oriented membrane multilayers have been examined. The copper X-ray absorption edge spectra indicate the presence of a tetragonal copper, which we assign as CuB, oriented with the long axis approximately orthogonal to the membrane normal. We have also detected the presence of a relatively long (2.6 A) Cu-S or Cu-Cl interaction, which we assign to a copper-thioether (probably Met210) coordination at the CuA site, with the bond oriented along the membrane normal. The coordination of the zinc, the iron and the CuB heme a3 binuclear site are discussed.

Animals↗
PubMed1993

The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation.

All photosynthetic organisms, with the exception of several species of photosynthetic bacteria, are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol. The association of this lipid with photosynthetic membranes has led to the assumption that it plays some role in photosynthesis. Stable null mutants of the photosynthetic bacterium Rhodobacter sphaeroides completely lacking sulfolipid were obtained by disruption of the sqdB gene. The ratios of the various components of the photosynthetic electron transport chain, as well as the electron transfer rates during cyclic electron transport, were not altered in the mutants, when grown under optimal conditions. Growth rates of wild type and mutants were identical under a variety of growth conditions, with the exception of phosphate limitation, which resulted in reduced growth of the mutants. Phosphate limitation of the wild type caused a significant reduction in the amount of all phospholipids and an increased amount of sulfolipid. By contrast, the sulfolipid-deficient mutant had reduced levels of phosphatidylcholine and phosphatidylethanolamine but maintained a normal level of phosphatidylglycerol. In addition, two unidentified lipids lacking phosphorus accumulated in the membranes of both wild-type and mutant strains under phosphate limitation. We conclude that sulfolipid plays no significant unique role in photoheterotrophic growth or photosynthetic electron transport in R. sphaeroides but may function as a surrogate for phospholipids, particularly phosphatidylglycerol, under phosphate-limiting conditions.

Bacterial Chromatophores↗
PubMed1993

Petroleum spill bioremediation in marine environments.

Bioremediation is a promising technology for responding to marine oil spills. A majority of molecules in crude oils and refined products are biodegradable, and they will eventually leave the environment as they are consumed by microbes. Bioremediation aims to stimulate the rate of this process. Successful bioremediation efforts have so far focused on applying fertilizers to aerobic oiled shorelines to at least partially relieve the nitrogen limitation of biodegradation by indigenous microorganisms. Nevertheless, there seems to be room for improving the process by developing better fertilizers, developing surfactants to stimulate degradation, and perhaps using exogenous bacteria. There also is room to extend the application to oiled marshes and other anaerobic sediments, and perhaps to floating slicks. This review covers our present understanding of hydrocarbon degradation in the marine environment, and discusses field trials and field use of bioremediation as an important adjunct to other tools for responding to marine oil spills.

Biodegradation, Environmental↗
PubMed1992

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.

Circular Dichroism↗
PubMed1992

Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus.

By deletion of the pufX gene of Rhodobacter capsulatus from a plasmid carrying the puf operon and complementation of a chromosomal puf operon deletion, we created pufX mutants and used them to characterize possible functions of the pufX gene product. The pufX mutants were incapable of photosynthetic growth in a minimal medium, or in a rich medium at low light intensities, although second-site mutations suppressed this phenotype. Measurements made in vitro with intact and solubilized chromatophore preparations indicated that the individual complexes of the photosynthetic unit seemed to function normally, but electron transfer from the reaction center to the cytochrome b/c1 complex was impaired. The structures of the photosynthetic apparatus of pseudo-wild type and mutant strains were evaluated using absorption spectroscopy and electron microscopy. The pufX mutants had intracytoplasmic membrane invaginations about 50% larger in diameter than those of the pseudo-wild type and higher levels of B870 light-harvesting complex. It is concluded that the PufX protein plays an important role in the structure of the functional photosynthetic unit, and its absence results in loss of efficient electron transfer from the QB site of the reaction center to the Qz site of the cytochrome b/c1 complex.

Base Sequence↗