Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

THE MECHANISM OF ACTION OF COLCHICINE : Colchicine Binding Properties of Sea Urchin Sperm Tail Outer Doublet Tubulin

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t_½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t_½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 10⁵ liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (K_i = 1.3 x 10^-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules.     

Thermodynamic resolution of the iron-sulfur centers of the succinic dehydrogenase of Rhodopseudomonas sphaeroides.

A single alkaline wash removes most of the succinic dehydrogenase activity from chromatophores of Rhodopseudomonas sphaeroides. Three iron-sulfur centers are also removed by this washing. Two of these are ferredoxin-like centers with electron paramagnetic resonance signals at g_v = 1.94 and midpoint potentials of +50 and ?250 mV at pH 7. The third is a high-potential iron-sulfur protein type signal centered at g 2.01 and a midpoint potential of +80 mV at pH 7. These centers have very similar properties to those of the well-characterized mammalian succinic dehydrogenase and account for the majority of iron-sulfur centers observed in chromatophores. Because it is so easily removed, it is concluded that succinic dehydrogenase is located on the outer surface of the chromatophore membrane, a conclusion supported by the fact that removal of the enzyme does not interfere with the kinetics of light-induced electron flow, nor does it allow cytochrome c₂ to escape from inside the chromatophore vesicles.     

Effect of oxidation-reduction potential on light-induced cytochrome and bacteriochlorophyll reactions in chromatophores from the photosynthetic green bacterium Chlorobium

The reaction center bacteriochlorophyll of Chlorobium thiosulfatophilum has a midpoint oxidation-reduction potential (E_m) of +330 mV. Its photooxidation is unaffected by oxidation-reduction potentials in the range from +260 mV to ?70 mV but on further reduction is attenuated to zero in a one-electron transition with an E_m of ?130 mV.A c-type cytochrome with an E_m of +220 mV and absorption maxima at 551–552 nm (α-band) and 420 nm (γ-band) is present in Chlorobium chromatophores and undergoes photooxidation. Cytocrome c photooxidation is attenuated to zero in two 1-electron steps with E_m of +30 mV and ?130 mVPossible roles for +30 mV and ?130 mV components in photosynthetic electron transport in Chlorobium are discussed.     

Fluorescence lifetime spectra of in vivo bacteriochlorophyll at room temperature

Fluorescence lifetime spectra of Rhodopseudomonas sphaeroides chromatophores have been measured at room temperature by phase fluorimetry at 82 MHz in order to investigate the heterogeneity of the emission. The total fluorescence was decomposed into two main components. A constant component, F_c, centered at 865 nm, represents about 50% of the total emission from dark-adapted chromatophores (F_o) and has a lifetime of 0.55 ns. A variable component is centered at 890 nm. Upon closing the reaction centers, 5-fold increases take place in both emission yield and lifetime of this component. In the dark-adapted state, its lifetime is about 50 ps and its contribution to the total fluorescence is 70% at 890 nm. In the presence of sodium dithionite, a long-lifetime component ($\text{τ}{}_{\mathrm{<mtext>D</mtext>}}\text{? 4}\text{ns}$) is observed. This probably arises from radical pair recombination between P⁺ and I^? (P, the primary electron donor, is a dimer of bacteriochlorophyll; I, the primary electron acceptor, is a molecule of bacteriopheophytin). Its spectrum is nearly identical to that of the variable component. This emission seems to be present also under nonreducing conditions, although with a much weaker intensity than when the electron acceptor quinone is prereduced.     

Effects of electron donors and acceptors on the kinetics of the photoelectric responses in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores

Chromatophores of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides were adhered to one side of a collodion film impregnated with a phospholipid solution in decane and 20 ns laser flashes were delivered to produce an electrical potential difference generated across the collodion film in less than 0.2 μs (resolution time of the apparatus). The kinetics of Δψ decay in the dark was studied. In the absence of additions there occurs a ‘rapid’ decay of photoelectric potential (τ ≈ 70 ms) corresponding to charge recombination within the primary dipole P-870⁺-Q^-_A. The rapid decay of Δψ is prevented by ascorbate in the presence of permeable redox dyes which can reduce the photooxidized P-870⁺ rapidly. Under these conditions, Δψ dissipates with τ > 0.5 s typical of a passive discharge of the chromatophore membrane. Prevention of the rapid decay of Δψ by 70–75% can be observed upon addition of excess ubiquinone-10 to the solution of phospholipids used to impregnate the collodion film, and to a lesser extent by addition of some other quinones. The effect of quinones is inhibited by o-phenanthroline. The data obtained show that upon association of chromatophores with the collodion film, the secondary quinone acceptor is extracted from its binding site into a hydrophobic volume of the macroscopic membrane, and this effect can be reversed by exogenous ubiquinone. About 4-times less Q-10 is required to reconstitute Q_B function in chromatophores from Rps. sphaeroides than in those from R. rubrum, which points to a tighter binding of the secondary acceptor in the former. No evidence for electrogenic nature of Q⁻_A → Q_B electron transfer could be obtained in experiments with Q_B-replenished chromatophores.     

Synthesis and possible character of a high-energy intermediate in bacterial photophosphorylation

1. In photophosphorylation with chromatophores from Rhodospirillum rubrum, evidence is presented for the synthesis of activated precursors of ATP in the energy-conversion system coupled to photosynthetic electron transport. 2. A significant amount of ATP is synthesized when a reaction mixture containing chromatophores and ADP is illuminated and then incubated with P_i in the dark. ATP is not synthesized to an appreciable extent, either when a reaction mixture containing chromatophores and P_i is illuminated and then incubated with ADP in the dark, or when one containing chromatophores alone is illuminated and then incubated with ADP and P_i in the dark. The amount of ATP thus synthesized is influenced markedly by concentrations of ADP. 3. The chromatophores illuminated with ADP, if allowed to stand in the dark at 30°, gradually lose the ability to form ATP with P_i in the dark. No loss of the ability occurs when the chromatophores illuminated with ADP are allowed to stand in the dark at 13° or in a frozen state. 4. Mg²⁺ is absolutely required for chromatophores to form ATP in the dark after illumination in the presence of ADP, and for the chromatophores to achieve ATP formation with P_i in the dark. 5. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide and o-phenanthroline strongly inhibit the light-dependent acquisition of the ability to form ATP with P_i in the dark, but not the consequent ATP formation with P_i in the dark. Arsenate, 2,4-dinitrophenol, quinacrine hydrochloride, quinine hydrochloride and pyrophosphate inhibit the former or the latter, or both. Oligomycin inhibits the former somewhat more than the latter. 6. From these findings it is suggested that a high-energy intermediate is formed in photosynthetic ATP formation, and that its formation is dependent on ADP but not P_i.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

Ion-dependent Inactivation of Barium Current through L-type Calcium Channels

It is widely believed that Ba²⁺ currents carried through L-type Ca²⁺ channels inactivate by a voltage- dependent mechanism similar to that described for other voltage-dependent channels. Studying ionic and gating currents of rabbit cardiac Ca²⁺ channels expressed in different subunit combinations in tsA201 cells, we found a phase of Ba²⁺ current decay with characteristics of ion-dependent inactivation. Upon a long duration (20 s) depolarizing pulse, I_Ba decayed as the sum of two exponentials. The slow phase (τ ≈ 6 s, 21°C) was parallel to a reduction of gating charge mobile at positive voltages, which was determined in the same cells. The fast phase of current decay (τ ≈ 600 ms), involving about 50% of total decay, was not accompanied by decrease of gating currents. Its amplitude depended on voltage with a characteristic U-shape, reflecting reduction of inactivation at positive voltages. When Na⁺ was used as the charge carrier, decay of ionic current followed a single exponential, of rate similar to that of the slow decay of Ba²⁺ current. The reduction of Ba²⁺ current during a depolarizing pulse was not due to changes in the concentration gradients driving ion movement, because Ba²⁺ entry during the pulse did not change the reversal potential for Ba²⁺. A simple model of Ca²⁺-dependent inactivation (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1993. J. Gen. Physiol. 102:1005–1030) robustly accounts for fast Ba²⁺ current decay assuming the affinity of the inactivation site on the α₁ subunit to be 100 times lower for Ba²⁺ than Ca²⁺.     

The Structure of Rhodospirillum rubrum

Cells from serial cultures of R. rubrum, grown anaerobically in the light, were harvested at intervals from ½ to 15 days and sectioned for electron microscopy by conventional methods. Cells of this species possess a multilayered outer envelope, and the external cell surface is differentiated into ridges extending parallel or obliquely to the long axis of the cell. Cells from very young cultures resemble non-photosynthetic bacteria and contain only a granular cytoplasm, scattered high-density particles, and low-density areas corresponding to the chromatin areas observed by light microscopy. They contain neither the chromatophores nor the lamellar systems assumed by previous investigators to be characteristic of this species when grown anaerobically in the light. Chromatophores appear in cells from cultures older than about 12 hours, while systems of paired lamellae appear along with the chromatophores in cells from cultures older than about 8 days. Divergent opinions concerning the occurrence of chromatophores or lamellae in this species can be resolved on the basis of the age of cultures used in previous studies. Other changes occurring in cells from cultures of increasing age include the appearance of granular and reticulate cytoplasmic bodies and vacuoles, extension of the chromatin areas, and the appearance of a single membrane enclosing several chromatophores.     

A Nuclear Magnetic Resonance Study of Water in Cold-acclimating Cereals

Continuous wave nuclear magnetic resonance (NMR) studies indicated that the line width of the water absorption peak (Δv½) from crowns of winter and spring wheat (Triticum aestivum L.) increased during cold acclimation. There was a negative correlation between Δv½ and crown water content, and both of these parameters were correlated with the lowest survival temperature at which 50% or more of the crowns were not killed by freezing (LT₅₀). Regression analyses indicated that Δv½ and water content account for similar variability in LT₅₀. Slow dehydration of unacclimated winter wheat crowns by artificial means resulted in similarly correlated changes in water content and Δv½. Rapid dehydration of unacclimated crowns reduced water content but did not influence Δv½. The incubation of unacclimated winter wheat crowns in a sucrose medium reduced water content and increased Δv½. The increase in Δv½ appears to be dependent in part on a reduction in water content and an increase in solutes.     

Photoelectric effects in bacterial chromatophores. Comparison of spectral and direct electrometric methods

Generation of photoelectric potential in chromatophores of Rhodopseudomonas sphaeroides has been measured (i) spectrophotometrically, using electrochromic shift of carotenoid absorption band or (ii) electrometrically, by means of two electrodes separated by a collodion film covered on one side with chromatophores. A 15 ns laser flash was used to induce a single turnover of photosynthetic reaction centers. It was found that results obtained by both methods are similar in (i) direction of electric vector (the chromatophore interior positive) and (ii) redox titration curves (E_m = 10mV). The magnitudes of the photopotential were about 60 and 25 mV, when monitored with spectral and electrometric techniques, respectively. In both cases, the rise times of the photopotentials were faster than time resolution of the techniques used. Decay of the response of carotenoids was found to be slower than that in the collodion film system. The addition of ubiquinone Q₁₀ into the decane solution of asolectin used to impregnate the collodion film led to slowing down of the decay. The carotenoid response decay could be accelerated by FCCP or o-phenanthroline. In the latter case, the shape of the decay curve coincides with decay of the photopotential measured in the collodion film system. It is suggested that decane extracts secondary ubiquinone from chromatophores attached to the collodion film. Such an unfavorable effect can be strongly decreased by added ubiquinone     

Photosynthetic Adaptation by Synechococcus leopoliensis in Response to Exogenous Dissolved Inorganic Carbon

Synechococcus leopoliensis was grown over a wide range of dissolved inorganic carbon (DIC) concentrations (4-25,000 micromolar) which were obtained by varying culture pH (6.2-9.6) and the CO₂ concentration of the gas stream (36-50,000 microliters per liter). The [DIC] required to half-saturate photosynthesis (K_½^DIC) was found to vary depending upon the ambient DIC concentration at which the cells were grown. Low [DIC] grown cells exhibited low values of K_½^DIC (4.7 micromolar) whereas cells grown at high [DIC] exhibited high values of K_½^DIC (1-2.5 millimolar). Intermediate concentrations of DIC produced intermediate values. Changes in K_½^DIC appeared to be solely a function of [DIC] and were independent of both culture pH and CO₂ concentration. As changes in K_½^DIC occur in response to DIC concentrations commonly found in natural systems we suggest this adaptation may be of ecological significance.     

Turnover and uptake by organs of radioactive serum high-density lipoprotein cholesteryl esters and phospholipids in the rat in vivo

The serum decay of rat serum high-density lipoprotein (HD lipoprotein), labelled biosynthetically with ³²P in the phospholipid or with ³H in the cholesteryl ester moiety, was measured in rats after partial hepatectomy or sham operation. The serum decay of ³H-labelled HD lipoprotein cholesteryl esters was biexponential. In sham-operated rats the t_½ values for the rapid phase and the slow phase were 0.2±0.1h and 4.2±0.4h (means±s.e.m.) respectively. After removal of two-thirds of the liver the t_½ value of the rapid phase did not change (0.1±0.1h), whereas the t_½ value of the slow phase increased to 5.7±0.8h. Partial hepatectomy hardly changed extrahepatic tissue radioactivities, whereas the percentage of the injected dose recovered in the liver 6h after injection decreased from 34.0±1.9% before to 13.5±1.6% after partial hepatectomy. The ³²P-labelled HD lipoprotein phospholipids showed a rapid monoexponential decay from serum with t_½ values of 0.71±0.3h and 1.48±0.11h after sham operation or partial hepatectomy respectively. The tissue ³²P radioactivities in the shamoperated rats, measured 1h after injection, were 46.0±1.7% (liver), 1.7±0.3% (adipose tissue), 3.7±1.2% (skeletal muscle) and 3.0±0.0% (erythrocytes) of the injected dose. Only the value for liver was affected by partial hepatectomy and decreased to 16.7±3.8%. In a previous publication [Van Tol, Van Gent, Van't Hooft & Vlaspolder (1978) Atherosclerosis 29, 439–448] we showed in a highly comparable experimental setting that the turnover rates of HD apolipoproteins A and C in vivo are not influenced by removal of two-thirds of the liver. From the present study it is clear that the removal rates of radioactive HD lipoprotein cholesteryl esters and HD lipoprotein phospholipids from serum in vivo are decreased by partial hepatectomy. The results indicate the possibility of partly separate metabolic pathways of HD apolipoproteins A and C, HD lipoprotein cholesteryl esters and HD lipoprotein phospholipids. The phospholipids and cholesteryl esters of HD lipoprotein are metabolized predominantly by the liver. Possible mechanisms for the hepatic uptake and metabolism of HD lipoprotein cholesteryl (esters) and phospholipids are discussed.     

Modification of room-temperature picosecond chlorophyll fluorescence kinetics in Photosystem-II-enriched particles by photochemistry

Room-temperature single photon timing measurements on Photosystem-II (PS II-) enriched thylakoid fragments at low excitation energies indicate the presence of three kinetic decay components of chlorophyll fluorescence arising from PS-II-associated pigments. Closing the PS II reaction centres produced three variable components, with lifetime values of 0.02–0.25, 0.15–0.90 and 0.35–2.0 ns, between the initial (F₀) and maximal (F_m) fluorescence levels. The yield of each component paralleled the changes in their respective lifetimes, indicating the presence of well-connected PS II reaction centres favouring energy transfer between each other. These changes show that variable chlorophyll fluorescence (F_v) does not arise from one specific origin. The extent of the modifications and the observed relationship between component lifetime and yield, on closing PS II reaction centres, cannot be explained by either the delayed fluorescence (charge recombination) hypothesis of Klimov and co-workers (Klimov, V.V. et al. (1978) Dokl. Akad. Nauk. SSSR 242, 1204–1207) or the proposed changes and origins put forward by Holzwarth and co-workers (Holzwarth, A.R. (1986) Photochem. Photobiol. 43, 707–725; Holzwarth, A.R. et al. (1985) Biochim. Biophys. Acta 807, 155–167).     

Kinetics of the Removal of Iron Pyrite from Coal by Microbial Catalysis

Different strains of Thiobacillus ferrooxidans and Thiobacillus thiooxidans were used to catalyze the oxidative dissolution of iron pyrite, FeS₂, in nine different coal samples. Kinetic variables and parametric factors that were determined to have a pronounced effect on the rate and extent of oxidative dissolution at a fixed Po₂ were: the bacterial strain, the nitrogen/phosphorus molar ratio, the partial pressure of CO₂, the coal source, and the total reactive surface area of FeS₂. The overall rate of leaching, which exhibited a first-order dependence on the total surface area of FeS₂, was analyzed mathematically in terms of the sum of a biochemical rate, ν₁, and a chemical rate, ν₂. Results of this study show that bacterial desulfurization (90 to 98%) of coal samples which are relatively high in pyritic sulfur can be achieved within a time-frame of 8 to 12 days when pulp densities are ≤20% and particle sizes are ≤74 μm. The most effective strains of T. ferrooxidans were those that were isolated from natural systems, and T. ferrooxidans ATCC 19859 was the most effective pure strain. The most effective nutrient media contained relatively low phosphate concentrations, with an optimal N/P molar ratio of 90:1. These results suggest that minimal nutrient additions may be required for a commercial desulfurization process.     

Changes from High Potassium (HK) to Low Potassium (LK) in Bovine Red Cells

Red cells of newborn calves contain 105–110 mmole K⁺ and 1–5 mmole Na⁺ per liter of cells. As the animals age the K⁺ content decreases to a value of 25–30 mmole/liter of cells after about 60 days. At approximately the same time, the sodium content reaches a value of 60–70 mmole/liter. The time required for half change (t_½) is 35–37 days for both Na⁺ and K⁺. The activity of (Na + K)-adenosine triphosphatase (ATPase) and the influx of K⁴² and Rb⁸⁶ into the red cells are high at birth and are reduced to 5 and 15% of their original values, respectively, in mature animals. t_½ for both is of the order of 30–35 days. The membrane Mg-ATPase activity is also high at birth and is reduced with a t_½ of 28–32 days to a final value of about 20% of its activity at birth. Separation of red cells according to their age showed that, in animals at the age of transition, newly formed red cells contain a higher K/Na ratio and a higher active transport capacity than older red cells of the same animal. It is suggested that the changes observed are a reflection of the average age of the red cell population as the animal grows.     

Strong orientational ordering of the near-infrared transition moment vectors of light-harvesting antenna bacterioviridin in chromatophores of the green photosynthetic bacterium Chlorobium limicola

The direction of the transition moments of chlorosome pigments in chromatophores of the green photosynthetic bacterium Chlorobium limicola was studied by linear dichroism. Orientation of chromatophores was achieved by stretching a polyacrylamide gel in which they were packed. It was shown that in each individual chromatophore the Q_y transition moment vectors of the whole chlorosome bacterioviridin are parallel to each other and are practically ideally oriented along the chlorosome long axis. The exact value of the angle α between the bacterioviridin transition moments and the long axis of the chlorosome is calculated to be α = 0°, the mean square deviation being 7°.     

The Respiratory Chain of Plant Mitochondria: XIV. Ordering of Ubiquinone, Flavoproteins, and Cytochromes in the Respiratory Chain

The effect of initial oxygen concentration on the rate and extent of oxidation of the respiratory chain carriers of anaerobic mitochondria from mung bean (Phaseolus aureus) seedlings was examined. The substrate was succinate, with malonate added to give malonate to succinate ratios of 6 to 12, thereby minimizing the flow of reducing equivalents from substrate and insuring maximal extent of oxidation of the carriers. The ratio of oxidizing equivalents available from oxygen to reducing equivalents available from reduced ubiquinone, designated the equivalents ratio, varied from 30 to 1. Cytochromes aa₃ and c₅₄₇ have unaltered oxidation half-times, designated t_{½ on}, as the equivalents ratio is reduced from 30 to 3, and the extent of oxidation is decreased by about 25%. The time of the oxidation-reduction cycle induced by the oxygen pulse, calculated from the point of half oxidation to that of half reduction and designated t_{½ off}, decreases 200 fold with this reduction in equivalents ratio. The oxidation half-time, t_{½ on}, for ubiquinone is unaltered by decreasing the equivalents ratio from 6 to 1; the value of t_{½ off} decreases only 30% while the extent of oxidation decreases 50%. The values of t_{½ on} and t_{½ off} and the extent of oxidation of cytochrome b₅₅₃ and flavoprotein Fp_ha were all much reduced at low equivalents ratios. The results, plus results from previous studies, indicate that there is the following linear sequence of components in the plant respiratory chain:     

Linkage disequilibrium in a finite population that is partially selfing

The linkage disequilibrium expected in a finite, partially selfing population is analyzed, assuming the infinite allele model. Formulas for the expected sum of squares of the linkage disequilibria and the squared standard linkage disequilibrium are derived from the equilibrium values of sixteen inbreeding coefficients required to describe the behavior of the system. These formulas are identical to those obtained with random mating if the effective population size N_e = (1-½S)N and the effective recombination value r_e = (1-S)r/(1-½S), where S is the proportion of selfing, are substituted for the population size and the recombination value. Therefore, the effect of partial selfing at equilibrium is to reduce the population size by a factor 1-½S and the recombination value by a factor (1-S)/(1-½S).