Biochemical and biophysical studies on cytochrome aa3 VIII. Effect of cyanide on the catalytic activity

1. 1. Cyanide inhibits the catalytic activity of cytochrome aa₃ in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·10³ M⁻¹·s⁻¹ and a K_i that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3.

2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa₃−O₂ system a biphasic reduction of cytochrome c occurs corresponding to an initial K_i of 0.8 μM and a final K_i of about 0.1 μM for the cytochrome aa₃−cyanide reaction.

3. 3. The inhibited species (a²+a₃³⁺HCN) is formed when a²⁺a₃³⁺ reacts with HCN, when a²⁺a₃²⁺HCN reacts with oxygen, or when a³⁺a₃³⁺HCN (cyano-cytochrome aa₃) is reduced. Cyanide dissociates from a²⁺a₃³⁺HCN at a rate of 2·10⁻³ s⁻¹ at 22 °C, pH 7.3.

4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a²⁺a₃³⁺ than to a³⁺a₃³⁺.

Rate of electron transfer between plastocyanin, cytochrome ƒ, related proteins and artificial redox reagents in solution

The rate of electron transfer between reduced cytochrome ƒ and plastocyanin (both purified from parsley) has been measured as k = 3.6 · 10⁷ M⁻¹ · s⁻¹, at 298 °K and pH 7.0, with activation parameters ΔH^‡ = 44 kJ · mole⁻¹ and ΔS^‡ = +46 J · mole⁻¹ · °K⁻¹. Replacement of cytochrome ƒ with red algal cytochrome c-553, Pseudomonas cytochrome c-551 and mammalian cytochrome c gave rates at least 30 times slower: k = 5 · 10⁵, 7.5 · 10⁵ and 1.0 · 10⁶ M⁻¹ · s⁻¹, respectively.

Similar measurements made with azurin instead of plastocyanin gave k = 6 · 10⁶ and approx. 2 · 10⁷ M⁻¹ · s⁻¹ for reaction of reduced azurin with cytochrome ƒ and algal cytochrome respectively.

Rate constants of 115 and 80 M⁻¹ · s⁻¹ were found for reduction of plastocyanin by ascorbate and hydroquinone at 298 °K and pH 7.0. The rate constants for the oxidation of plastocyanin, cytochrome ƒ, Pseudomonas cytochrome c-551 and red algal cytochrome c-553 by ferricyanide were found to be between 3 · 10⁴ and 8 · 10⁴ M⁻¹ · s⁻¹.

The results are discussed in relation to photosynthetic electron transport.     

Kinetics and equilibria of Ga(III)---thiocyanate complex formation. Mechanism of ligand substitution reactions of Ga(III) in aqueous solution

The kinetics and equilibria of complex formation by Ga(III) with NCS⁻ in aqueous solution have been measured over a range of acidities and temperatures, the contributing paths to the reaction resolved, and their rate constants and activation parameters determined. The hydrolysis equilibria required to carry out this resolution of kinetic behaviour have also been measured.

Unlike the other reported complexation reactions of Ga(III) in aqueous solution, the separate reaction pathways can be assigned with no ambiguity. At 25 °C and ionic strength 0.5 M, the observed forward rate constant for the complex formation is described by {k₁ + k₂K_1h/[H⁺] + k₃K_1hK_2h/[H⁺]²} M⁻¹ s⁻¹. For these conditions, the first and second successive hydrolysis constants of Ga(H₂O)₆³⁺ are given by pK_1h = 3.69 ± 0.01 and pK_2h = 3.74 ± 0.04. The rate constants corresponding to the reactions of the species Ga(H₂O)₆³⁺, Ga(H₂O)₅(OH)²⁺ and Ga(H₂O)₄(OH)₂⁺ with NCS⁻ are k₁ = 57 ± 4 M^{−1 −1}, k₂ = (1.08 ± 0.01) × 10⁵ M⁻¹ s⁻¹ and k₃ = 3 × 10⁶ M⁻¹ s⁻¹ respectively. The complexation equilibrium quotient [GaNCS²⁺]/([Ga³⁺][NCS⁻]) has been independently determined by spectrophotometric titration to be 20.8 ± 0.3 M⁻¹ at 25 °C and ionic strength 0.5 M.

These kinetic results lead to an interpretation of the data, and a reinterpretation of other data for aquo-Ga(III) complex formation kinetics from the literature which support the assignment of a dissociative interchange mechanism for these reactions rather than the associative activation mode sometimes proposed.     

Interaction of anthracycline antibiotics with biopolymers: 7. Equilibrium binding studies on the interaction of iremycin and DNA

We report spectrophotometric equilibrium studies of both the self-association of the new antibiotic iremycin and of its binding to calf thymus DNA in solution (ionic strength 0.2 M; pH 6.0). Iremycin forms dimers in this solution with a dimerization constant K₄=(1.19 ± 0.10) × 10³ M⁻¹. This equilibrium is taken into account in the evaluation of the interaction of iremycin with DNA. The binding behaviour can be completely described by a single binding mechanism of monomeric iremycin to DNA with allowance both for neighbour exclusion and for cooperativity of interaction. The three intrinsic binding parameters for the homogeneous model were determined simultaneously by a least squares fit of the original titration data: equilibrium constant of cooperative binding K = (2.72 ± 0.66) × 10⁵ M⁻¹ cooperativity parameter σ=0.38±3.27 ± 0.32. The binding parameters of iremycin and adriamycin and their microbial activities are compared.     

Structure of 7S seed globulin from Phaseolus vulgaris L. in solution

The structure of the 7S globulin from Phaseoulus vulgaris L in dilatue solutions has been studied by small angle X-ray scattering (SAXS), by quasi-elastic light scattering (Q ELS), by circular dichroism spectroscopy (c.d.), and by precise density measurements. The molar mass, the radius of gyration, the volume, the maximum dimension and the diffusion coefficient were determined as M = 1.45 × 10⁵ g mol⁻¹, R_G = 4.05 nm, V = 300- nm³, L = 13.0 nm and D_20,w⁰ = 4.5 × 10⁻⁷ cm² s⁻¹, respectively. The molecule has an asymmetrical shape with the dimensions 12.5 × 12.5 × 3.75 nm. The secondary structure of the 7S globulin is characterized by a small portion of -helical structure (14%) and a marked content of β-structure (18%).     

Kinetics and mechanism of the stoichiometric oxygenation of [Cu(fla)(idpa)]ClO4 [fla=flavonolate, IDPA=3,3′-imino-bis(N,N-dimethylpropylamine)] and the [Cu(fla)(idpa)]ClO4-catalysed oxygenation of flavonol

Oxygenation of [Cu^II(fla)(idpa)]ClO₄ (fla=flavonolate; IDPA=3,3′-iminobis(N,N-dimethylpropylamine)) in dimethylformamide gives [Cu^II(idpa)(O-bs)]ClO₄ (O-bs=O-benzoylsalicylate) and CO. The oxygenolysis of [Cu^II(fla)(idpa)]ClO₄ in DMF was followed by electronic spectroscopy and the rate law −d[{Cu^II(fla)(idpa)}ClO₄]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂] was obtained. The rate constant, activation enthalpy and entropy at 373 K are k_obs=6.13±0.16×10⁻³ M⁻¹ s⁻¹, ΔH^‡=64±5 kJ mol⁻¹, ΔS^‡=−120±13 J mol⁻¹ K⁻¹, respectively. The reaction fits a Hammett linear free energy relationship and a higher electron density on copper gives faster oxygenation rates. The complex [Cu^II(fla)(idpa)]ClO₄ has also been found to be a selective catalyst for the oxygenation of flavonol to the corresponding O-benzoylsalicylic acid and CO. The kinetics of the oxygenolysis in DMF was followed by electronic spectroscopy and the following rate law was obtained: −d[flaH]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂]. The rate constant, activation enthalpy and entropy at 403 K are k_obs=4.22±0.15×10⁻² M⁻¹ s⁻¹, ΔH^‡=71±6 kJ mol⁻¹, ΔS^‡=−97±15 J mol⁻¹ K⁻¹, respectively.     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Mitochondrial respiratory chain of Tetrahymena pyriformis The properties of submitochondrial particles and the soluble b and c type pigments

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 °C there are two b-type pigments with half-reduction potentials of −0.04 and −0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a₂ with E_m7.2 of 0.245 and 0.345 V.

EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spin ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g = 6, and a g = 2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed.

Cytochrome c₅₅₃ is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c₅₅₃ is a negatively charged protein at neutral pH with an E_m7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with a K_D of 0.8 · 10⁻⁶ M. Reduced cytochrome c₅₅₃ serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c₅₅₃ shows the presence of a low spin ferric heme with the dominant resonance signal at g = 3.28.

A pigment with an absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and E_m7.2 of −0.17 V and exhibits a high spin ferric heme signal at g = 6.     

Model reactions of a carbonylation catalyst: phosphite induced migratory CO insertion in [MeIr(CO)2I3]

Carbonylation of the anionic iridium(III) methyl complex, [MeIr(CO)₂I₃]⁻ (1) is an important step in the new iridium-based process for acetic acid manufacture. A model study of the migratory insertion reactions of 1 with P-donor ligands is reported. Complex 1 reacts with phosphites to give neutral acetyl complexes, [Ir(COMe)(CO)I₂L₂] (L = P(OPh)₃ (2), P(OMe)₃ (3)). Complex 2 has been isolated and fully characterised from the reaction of Ph₄As[MeIr(CO)₂I₃] with AgBF₄ and P(OPh)₃; comparison of spectroscopic properties suggests an analogous formulation for 3. IR and ³¹P NMR spectroscopy indicate initial formation of unstable isomers of 2 which isomerise to the thermodynamic product with trans phosphite ligands. Kinetic measurements for the reactions of 1 with phosphites in CH₂Cl₂ show first order dependence on [1], only when the reactions are carried out in the presence of excess iodide. The rates exhibit a saturation dependence on [L] and are inhibited by iodide. The reactions are accelerated by addition of alcohols (e.g. 18× enhancement for L = P (OMe)₃ in 1:3 MeOH-CH₂Cl₂). A reaction mechanism is proposed which involves substitution of an iodide ligand by phosphite, prior to migratory CO insertion. The observed rate constants fit well to a rate law derived from this mechanism. Analysis of the kinetic data shows that k₁, the rate constant for iodide dissociation, is independent of L, but is increased by a factor of 18 on adding 25% MeOH to CH₂Cl₂. Activation parameters for the k₁ step are ΔH^≠ = 71 (±3) kJ mol⁻, ΔS^≠ = −81 (±9) J mol⁻¹ K⁻¹ in CH₂Cl₂ and ΔH^≠ = 60(±4) kJ mol⁻¹, ΔS^≠ = −93(± 12) J mol⁻¹ K⁻¹ in 1:3 MeOH-CH₂Cl₂. Solvent assistance of the iodide dissociation step gives the observed rate enhancement in protic solvents. The mechanism is similar to that proposed for the carbonylation of 1.     

Cytochrome oxidase from Pseudomonas aeruginosa. IV. Reaction with oxygen and carbon monoxide

The reaction between a cytochrome oxidase from Pseudomonas aeruginosa and oxygen has been studied by a rapid mixing technique. The data indicate that the heme d₁ moiety of the ascorbate-reduced enzyme is oxidized faster than the heme c component. The oxidation of heme d₁ is accurately second order with respect to oxygen and has a rate constant of 5.7 · 10⁴ M⁻¹ · s⁻¹ at 20 °C. The oxidation of the heme c has a first-order rate constant of about 8 s⁻¹ at infinite concentration of O₂. The results indicate that the rate-limiting step is the internal transfer of electrons from heme c to heme d₁. These more rapid reactions are followed by more complicated but smaller absorbance changes whose origin is still not clear.

The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 · 10⁴ M⁻¹ · s⁻¹. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 · 10⁻⁶ M. From these data a rate constant of 0.041 s⁻¹ can be calculated for the dissociation of CO from the enzyme.     

Structural differences between ribosomes of various eukaryotes: stability, density, mass, size and structure in solution of cytoplasmic ribosomes from Tetrahumena, Artemia and Euglena

Cytoplasmic ribosomal particles were isolated from the ciliated protozoa Tetrahymena pyriformis, the brine shrimp Artemia, and the flagellated alga Euglena gracilis; their stability, density, mass, size and structure in solution have been compared in identical conditions. The small and in particular the large subunits of Euglena ribosomes were less stable than the corresponding particles of the other eukaryotes. Equilibrium centrifugation of the ribosomes of the various species in CsCl density gradients revealed no difference in either their homogeneity or their buoyant density; so no differences were detected in this way in the ratio of their RNA to protein content or in their partial specific volume. Using analytical boundary sedimentation, the sedimentation coefficients s_20,w⁰ of the whole ribosomes were measured to be 81, 81 and 86 S, respectively. Photon correlation spectroscopy of laser light scattered by the whole ribosomes has yielded values for their diffusion coeffcient D_20,w⁰) of 1.51, 1.42 and 1.29 × 10⁻⁷ cm² s⁻¹, respectively; this corresponds to values for their hydrodynamic radius of 142, 151 and 166 Å, respectively. The data indicate also that the solvation of the ribosomes increases with their size and mass.So evidence has been obtained for differences in the size of various eukaryotic ribosomes, and even in the degree of compactness of their structure in solution.     

Reduction of ferricytochrome c by hydrogen atoms: Evidence for intramolecular transfer of reducing equivalent

Direct evidence obtained by means of the technique of pulse radiolysis-kinetic spectrometry, with measurements in the time range 10⁻⁶ to 1 s, is presented that, consequent upon reaction of a single H-atom with a single molecule of ferricytochrome c, a reducing equivalent is transmitted via the protein structure to the ferriheme moiety. Such transmission accounts for at least 70% of the total reduction of the ferri to the ferro state of cytochrome c. The remainder of the total reduction takes place without stages resolvable on the time scale of these experiments. Reduction brought about by H atoms appears to follow a different course than reduction by hydrated electrons. In the latter case, intramolecular transmission of reducing equivalents could not be demonstrated (Lichtin, N. N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117–135).

Not every H-atom reacts with ferricytochrome c at a site which results in conversion of the Fe(III) state to the Fe(II) state. Approximately half of reacting H-atoms do not produce reduction.

The following second order rate constants have been determined in solutions of low ionic strength at 20±2 °C: k[H+ferricytochrome c] = (1.0±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0 and 6.7; k[H+ferrocytochrome c] = (1.3±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0; k[e⁻_aq + ferrocytochrome c] = (1.9±0.4) · 10¹⁰ M⁻¹ · s⁻¹ at pH 6.7.     

Biotransformation of monoterpenoids, (−)- and menthols, terpinolene and carvotanacetone by Aspergillus species

Four monoterpenoids, (−)- and (+)-menthols, terpinolene and carvotanacetone were biotransformed by Aspergillus niger and several related species. Aspergillus niger converted (−)-menthol to 1-, 2-, 6-, 7-, and 9-hydroxymenthols and the mosquito repellent-active 8-hydroxymenthol. On the other hand, (+)-menthol was smoothly biotransformed by A. niger to give 7-hydroxymenthol. Aspergillus cellulosae biotransformed (−)-menthol specifically to 4-hydroxymenthol. Terpinolene and (−)-carvotanacetone were converted by A. niger to two , β-unsaturated ketones, a fenchane-type compound and diastereoisomeric p-menthane-2,9-diols and 8-hydroxycarvomenthol, respectively.     

Kinetics of some electron-transfer reactions in biological Photosystems II. Study of intramolecular electron-transfer rates between ferredoxin-NADP reductase, NADP radical and oxidized ferredoxin

It has been shown by the pulse radiolysis technique that radiation-generated NADP free radicals (NADP·) first combine with ferredoxin-NADP reductase and then transfer the odd electron by a fast intramolecular process to the enzyme flavin moiety yielding the semiquinone (ferredoxin-NADP reductase, FNR-FADH·). The corresponding first-order rate constant k₁₅ varies with ionic strength from 2.6·10³ s⁻¹ at I = 0.66 M to 2.3·10⁴ s⁻¹ at I = 0.005 M In the presence of ferredoxin-NADP reductase-bound oxidized ferredoxin, the electron cascades, thus further reducing the ferredoxin. The transfer of the electron from the flavin semiquinone (ferredoxin-NADP reductase, FNR-FADH·) to the bound oxidized ferredoxin proceeds at a rate of k₁₈ = 2.36 s⁻¹. This process approaches an equilibrium condition which is in favor of the reverse reaction suggesting that k₋₁₈ > k₁₈.     

3nion-independent conformational ordering in iota-carrageenan: Disorder-order equilibria and dynamics

The equilibria and dynamics of the disorder-to-order transition of the anionic polysaccharide iota-carrageenan have been studied in the presence of tetramethyl-ammonium salts. By the use of a stopped-flow polarimeter, the rate equation and temperature dependence of the observed forward rate-constant were found to accord with a co-operative dimerisation process. Activation parameters for helix nucleation were shown to be independent of the anion for solutions containing tetramethylammonium chloride and bromide, i.e., ΔH^‡ = 1 ±3 kJ.mol⁻¹, ΔS^‡ = −178 ±10 J.mol⁻¹.K⁻¹, ΔG^‡_298K = 54 ±2 kJ.mol⁻¹, and k_nuc,298K = 1880 ±80 dm³.mol⁻¹.s⁻¹. The temperature dependence of optical rotation was also shown to be independent of the anion present.     

Rate constants and activation parameters for organo-cobalt porphyrin bond homolysis from NMR relaxation times

¹H NMR line broadening is found to be an effective complimentary method to chemical trapping for determining the rates and activation parameters for organo-metal bond homolysis events that produce freely diffusing radicals. Application of this method is illustrated by measurement of bond homolysis activation parameters for a series of organo-cobalt porphyrin complexes ((TPP)Co-C(CH₃)₂CN (ΔH^≠ = 19.5±0.9 kcal mol⁻¹, ΔS^≠ = 12±3 cal°K⁻¹ mol⁻¹), (TMP)Co-C(CH₃)₂CN (ΔH^≠ = 20±1 kcal mol⁻¹,ΔS^≠ = 13±2 cal°K⁻¹ mol⁻¹), (TAP)Co-C(CH₃)₂CO₂CH₃ (ΔH^≠ = 18.2±0.5 kcal mol⁻¹, ΔS^≠ = 12±2 cal °K⁻¹ mol⁻¹), (TAP)Co-CH(CH₃)C₆H₅ (ΔH^≠ = 22.5±0.5, ΔS^≠ = 17±2 cal °K⁻¹ mol⁻¹)). The line broadening method is particularly useful in determining activation parameters for dissociation of weakly bonded organometallics where the rate of homolysis can exceed the range measurable by conventional chemical trapping methods.     

Bioaccumulation of copper(II), lead(II) and chromium(VI) by growing Aspergillus niger

The effect of copper(II), lead(II) and chromium(VI) ions on the growth and bioaccumulation properties of Aspergillus niger was investigated as a function of initial pH and initial metal ion concentration. The optimum pH values for growth and metal ion accumulation were determined as 5.0, 4.5 and 3.5 for copper(II), lead(II) and chromium(VI) ions, respectively. Although all metal ion concentrations caused an inhibition effect on the growth of A. niger, it was capable of removing of copper(II) and lead(II) with a maximum specific uptake capacity of 15.6 and 34.4 mg g⁻¹ at 100 mg dm⁻³ initial copper(II) and lead(II) concentration, respectively. Growth of A. niger was highly effected by chromium(VI) ions and inhibited by 75 mg dm⁻³ initial chromium(VI) concentration since some inhibition occurred at lower concentrations.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data

High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg⁻¹, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg²⁺). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp³⁻ (ao) AMp²⁻ (ao)+ ATp⁻ (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol ⁻¹, ΔH° = −1.5 ± 1. 5 kJ mol ⁻¹, °S ° = −17 ± 5 J mol⁻¹ K⁻¹, and ACP_p°≈ = −46 J mo1l⁻¹ K⁻¹ at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg²⁺] = 1.35 × 10⁻³ mol kg⁻¹, and I = 0.23 mol kg⁻¹) the apparent equilibrium constant (K_A′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]²) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10⁻⁴ mol kg⁻¹ for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates.     

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.

Two independent sets of measurements using a variety of methods show that a sample with A_{800 nm} = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.

Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.

These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: _{802 nm} = 288 (± 14) mM⁻¹ · cm⁻¹ and _{865 nm} = 128 (± 6) mM⁻¹ · cm⁻¹. For reversible bleaching of the 865-nm band, Δ^{red - ox}_865nm = 112 (± 6) mM⁻¹ · cm⁻¹ (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.