Search PubMed⌕ Search

Biomedical subjects

R C Prince

Publications and source records attributed to R C Prince.

At least 73 records · Page 4Linked to original sources
PubMed1987

On the role of the light-harvesting B880 in the correct insertion of the reaction center of Rhodobacter capsulatus and Rhodobacter sphaeroides.

The purple, non-sulfur photosynthetic bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides have two types of pigment-protein complexes that absorb incident light and funnel it to the photochemical reaction center. One of these, B880, is present at an essentially constant ratio to the reaction center, while the abundance of the other, B800-850, varies with growth conditions. Independent work in our two laboratories has indicated that while the absence of B800-850 permits photosynthetic growth in both organisms, the lack of B880 produces a different phenotype in the two species. Thus R. sphaeroides is still photosynthetically competent when it lacks this complex, while R. capsulatus is not. This unanticipated difference in what appear to be very closely related organisms has caused us to reexamine the properties of the two mutants simultaneously, under identical conditions. We find that the original descriptions are indeed correct; the reaction center of R. capsulatus is not correctly inserted into the photosynthetic membrane in the absence of B880, while that of R. sphaeroides is.

Bacterial Proteins↗
PubMed1986

Radical-pair energetics and decay mechanisms in reaction centers containing anthraquinones, naphthoquinones or benzoquinones in place of ubiquinone.

In reaction centers from Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides), light causes an electron-transfer reaction that forms the radical pair state (P+I-, or PF) from the initial excited singlet state (P) of a bacteriochlorophyll dimer (P). Subsequent electron transfer to a quinone (Q) produces the state P+Q-. Back electron transfer can regenerate P from P+Q-, giving rise to 'delayed' fluorescence that decays with approximately the same lifetime as P+Q-. The free-energy difference between P+Q- and P can be determined from the initial amplitude of the delayed fluorescence. In the present work, we extracted the native quinone (ubiquinone) from Rps. sphaeroides reaction centers, and replaced it by various anthraquinones, naphthoquinones, and benzoquinones. We found a rough correlation between the halfwave reduction potential (E1/2) of the quinone used for reconstitution (as measured polarographically in dimethylformamide) and the apparent free energy of the state P+Q- relatively to P. As the E1/2 of the quinone becomes more negative, the standard free-energy gap between P+Q- and P decreases. However, the correlation is quantitatively weak. Apparently, the effective midpoint potentials (Em) of the quinones in situ depend subtly on interactions with the protein environment in the reaction center. Using the value of the Em for ubiquinone determined in native reaction centers as a reference, and the standard free energies determined for P+Q- in reaction centers reconstituted with other quinones, the effective Em values of 12 different quinones in situ are estimated. In native reaction centers, or in reaction centers reconstituted with quinones that give a standard free-energy gap of more than about 0.8 eV between P+Q- and P*, charge recombination from P+Q- to the ground state (PQ) occurs almost exclusively by a temperature-insensitive mechanism, presumably electron tunneling. When reaction centers are reconstituted with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 eV, part or all of the decay proceeds through a thermally accessible intermediate. There is a linear relationship between the log of the rate constant for the decay of P+Q- via the intermediate state and the standard free energy of P+Q-. The higher the free energy, the faster the decay. The kinetic and thermodynamic properties of the intermediate appear not to depend strongly on the quinone used for reconstitution, indicating that the intermediate is probably not simply an activated form of P+Q-.(ABSTRACT TRUNCATED AT 400 WORDS)

Anthraquinones↗
PubMed1986

Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation.

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.

Antimycin A↗
PubMed1986

Cytochrome c(2) is not essential for photosynthetic growth of Rhodopseudomonas capsulata.

The structural gene for cytochrome c(2) (cycA) of the photosynthetic bacterium Rhodopseudomonas capsulata has been cloned, and the nucleotide and deduced polypeptide sequences have been determined. Compared with the known amino acid sequence of the purified cytochrome c(2), the nucleotide sequence corresponding to the N-terminal part of the cycA gene product indicates the presence of a putative 21 amino acid signal sequence. Thus, cytochrome c(2) may be synthesized as a precursor which is processed during its secretion to the periplasm. Insertion and insertion-deletion mutations were constructed in vitro and the chromosomal cycA(+) allele of a wild-type strain was replaced with these mutations by homologous recombination to yield c(2) (-) mutants of R. capsulata. The c(2) (-) mutants are stable, and they can grow by photosynthesis and by respiration. Since cytochrome c(2) is the primary electron donor to the reaction center during photosynthesis, the ability of these mutants to grow photosynthetically indicates that an alternative way(s) of reducing the oxidized reaction center must exist in R. capsulata. One candidate for this role may be the membrane-bound cytochrome c(1).

Journal Article↗
PubMed1985

The effect of chloride on the redox and EPR properties of myeloperoxidase.

Myeloperoxidase was purified from human polymorphonuclear leukocytes and the effect of chloride upon the EPR and potentiometric properties was studied. The redox titration between the ferrous and ferric states of the enzyme yielded n = 1 Nernst plots between pH 9 and 4, with clear isosbestic points in the optical spectra during the redox change. The midpoint potential (Em) between the ferric and ferrous forms of the enzyme exhibited a pH-dependent change between pH 4 and 9, and the effect of added chloride ion indicated that Cl- competed with OH- for a binding site on the enzyme. Interestingly, the pH dependence of the Em indicated that the overall redox reactions of the enzyme was: ferric myeloperoxidase + 2e- + 1H+ = ferrous myeloperoxidase. Myeloperoxidase exhibited a rhombic high spin EPR signal which exhibited reduced rhombicity upon the binding of chloride. Our results strongly suggest that chloride binds to the sixth coordination position of the chlorin iron in myeloperoxidase by replacing the water which is the sixth ligand in the resting state. It is also concluded that the two iron centers are identical and that there is no interaction between them.

Binding, Competitive↗
PubMed1985

Methyl purple, an exceptionally sensitive monitor of chloroplast photosystem I turnover: physical properties and synthesis.

Methyl purple is a recently introduced quinone-imide redox dye that has been shown to be an exceptionally sensitive monitor of photosystem I activity in chloroplasts. This compound has a wide range of potential applications for studies of photosynthetic electron transfer and photosynthetic bioenergetics, but the physical properties of the compound must first be established rigorously. The specific molar absorption coefficients of both the anionic and protonated forms of methyl purple have been determined. The oxidation-reduction midpoint potential of methyl purple over the pH range 3 to 12 was also determined by polarographic methods, and the effect of pH on the visible absorption spectrum is reported. A detailed procedure for the synthesis of methyl purple is given.

Chemical Phenomena↗
PubMed1984

One- and two-electron reduction of hydroxy-1,4-naphthoquinones and hydroxy-9,10-anthraquinones. The role of internal hydrogen bonding and its bearing on the redox chemistry of the anthracycline antitumour quinones.

First and second half-wave reduction potentials of 11 1,4-naphthoquinones and 42 9,10-anthraquinones have been measured for solutions in dimethylformamide. The presence of hydroxy groups at the 5- and 8-positions of the 1,4-naphthoquinone nucleus, and at the 1-, 4-, 5- and 8-positions of the 9,10-anthraquinone (the alpha-positions) markedly raises both reduction potentials. Measurements on the corresponding methoxy- and acetoxyquinones indicate that internal hydrogen bonding in the alpha-hydroxyquinones makes a major contribution to stabilisation of the semiquinone, probably as a result of increased delocalisation due to exchange of the hydroxy hydrogen between the two neighbouring oxygen atoms. The bearing of this phenomenon on the mechanism of action of anthracycline antitumour quinones is discussed, and the stabilising effect on the semiquinone of hydroxy groups at the 1- and 5-positions of the 9,10-anthraquinone nucleus is highlighted.

Anthraquinones↗
PubMed1984

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems.

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.

Animals↗
PubMed1983

Resolution of the hemes of hydroxylamine oxidoreductase by redox potentiometry and optical spectroscopy.

Optical spectroscopy combined with redox potentiometry has resolved the hemes of hydroxylamine oxidoreductase into 6 thermodynamically distinct classes. There are apparently 4 classes of heme c553, with Em7-values of 295 mV, 10 mV, -190 mV and -390 mV, present in a stoichiometry of 1:1:2:1; two equivalents of heme c559, Em7 O mV, and one of heme P-460, an unusual chromophore, with Em7 -260 mV.

Heme↗
PubMed1982

Reduction of cytochromes b6 and f in isolated plastoquinol-plastocyanin oxidoreductase driven by photochemical reaction centers from Rhodopseudomonas sphaeroides.

Photochemical reaction centers isolated from the bacterium Rhodopseudomonas sphaeroides are able to donate electrons to cytochromes b6 and f in the plastoquinol,-platocyanin oxidoreductase isolated from spinach chloroplasts. The reduction reactions occur only after the second single turnover flash, in a reaction which is sensitive to inhibitors of the reactions in the chloroplast membranes. When all the components of the b6f complex are oxidized prior to activation, both cytochromes b6 and f are reduced after the second flash. If cytochrome f is reduced prior to activation, cytochrome b6 is still reduced after the second flash, but as the potential is lowered so that the Rieske iron-sulfur cluster is reduced prior to activation, the reduction of cytochrome b6 fails. The b6f complex thus functions in such a way that a two-electron redox couple, probably a quinone, is capable of reducing both cytochrome b6 and cytochrome f, the latter via the Rieske iron-sulfur cluster, in a coupled reaction where both electrons must leave the two-electron carrier. Cytochrome b6 is thus reduced in a manner analogous to "oxidant-induced reduction."

Bacterial Proteins↗
PubMed1981

The thermodynamic properties of some commonly used oxidation-reduction mediators, inhibitors and dyes, as determined by polarography.

The oxidation-reduction midpoint potentials (Em) of the following compounds have been measured in the range of pH from 3 to 12 by polarography: methyl viologen; benzyl viologen; 2-hydroxy-1,4-naphthoquinone; 2-hydroxy-1,4-anthraquinone; N,N,N',N',-tetramethyl-p-phenylenediamine; 2,3,5,6-tetramethyl-p-phenylenediamine; phenazine; N-methylphenazonium methosulfate; N-methylphenazonium sulfonate methosulfate; N-ethylphenazonium ethosulfate; pyocyanine; neutral red; safranin; phenol red; chlorophenol red; cresol red; bromocresol purple; 2,5-dibromo-3-methyl-6-isopropylbenzoquinone and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Many of these previously assumed to have a simple behavior in this range have proven to be rather more complicated, and several anomalous observations have been reconciled.

Dibromothymoquinone↗
PubMed1980

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides.

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c2, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation (t 1/2 3-5 microseconds). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (102 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Qz (QzH2) reduces the Rieske Center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c2 reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center. UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from gy 1.90 to gy 1.89, and shifts the Em,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c2, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of HII(+); these may reflect additional sites of action of the inhibitor.

Antimycin A↗
PubMed1980

The orientation of the magnetic axes of the membrane-bound iron-sulfur clusters of spinach chloroplasts.

Spinach chloroplast membranes were oriented onto mylar sheets by partial dehydration, and the orientation of the magnetic axes of membrane-bound paramagnetic clusters determined by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the reduced Rieske iron-sulfur cluster signal is of orthorhombic symmetry oriented with th gy = 1.90 axis orthogonal to the membrane plane and with the gz = 2.03 axis in the membrane plane; the gx-axis is undetectable, presumably due to its broadness. If the Rieske center is a two-iron iron-sulfur cluster, we conclude that the iron-iron axis lies in the plane of the membrane. Illumination reduces the two bound chloroplast iron-sulfur proteins known as Clusters A and B. Center A is oriented such that gx = 1.86 and gy = 1.94 lie at an angle of about 40, and gz = 2.05 is at approximately 25, to the membrane plane. There are two possible orientations of Cluster B depending on the set of g-values assigned to this cluster. For one set of g-values, gz = 2.04 and gx = 1.89 are oriented in the plane of the membrane while gy = 1.92 is orthogonal to the plane. Alternatively, gz = 2.07 and gy = 1.94 are oriented approximately 50 and 40 to the membrane plane respectively, and gx = 1.80 is in the plane of the membrane. An additional light-induced signal at g = 2.15 oriented orthogonal to the plane is currently unexplained, as are other membrane perpendicular signals seen at g = 2.3 and g = 1.73 in dark-adapted samples.

Cell Membrane↗
PubMed1979

The recognition of a special ubiquinone functionally central in the ubiquinone-cytochrome b-c2 oxidoreductase.

Although the energy conserving membranes of the photosynthetic bacterium Rhodopseudomonas sphaeroides contain a 25 (+/- 3)-fold molar excess of ubiquinone over the photochemical reaction center, the activity of the ubiquinone-cytochrome b-c2 oxidoreductase is unaffected by quinone extraction until only 3, or at most 4, ubiquinones remain; only then does further extraction prevent the function of the oxidoreductase. Since 2 of these last ubiquinones are integral parts of the photochemical reaction center, we conclude that the ubiquinone-cytochrome b-c2 oxidoreductase requires only 1, or at most 2, molecules of ubiquinone-10 for its function. Earlier kinetic data identified a major electron donor to ferricytochrome c2 as a single molecule (known as Z) which requires 2 electrons and 2 protons for its equilibrium reduction. Hence, we identify a single molecule of quinone, probably ubiquinone-10 in a special environment, as a major electron donor to ferricytochrome c2 in the ubiquinone cytochrome b-c2 oxidoreductase.

Cytochrome c Group↗
PubMed1979

Equilibrium and kinetic measurements of the redox potentials of cytochromes c2 in vitro and in vivo.

The equilibrium oxidation-reduction mipoint potential (Em) of isolated Rhodopseudomonas sphaeroides cytochrome c2 exhibits a pH-dependent behavior which can be ascribed to a pK on the oxidized form at pH 8.0 (Pettigrew et al. (1975) Biochim. Biophys. Acta 430, 197-208). However, as with mammalian cytochrome c (Brandt, K.G. Parks, P.C., Czerlinski, G.H. and Hess, G.P. (1966) J. Biol. Chem. 241, 4180-4185) this pK can more properly be attributed to the combination of a pK beyond pH 11, and a slow conformational change of the ferricytochrome. This has been demonstrated by resolving the Em of cytochrome c2 before and after the conformational change. The Em of the unaltered form is essentially pH independent between pH 7 and 11.5, and the lower equilibrium Em is due solely to the conformational change. In vivo the conformational change is prevented by the binding of the cytochrome c2 to the photochemical reaction center, and the cytochrome exhibits an essentially pH-independent Em from pH 5 to 11. The alkaline transition thus has little physiological significance, and it is unlikely that the redox reactions of cytochrome c2 in vivo involve protons.

Cytochrome c Group↗