Probing fluorescence induction in chloroplasts on a nanosecond time scale utilizing picosecond laser pulse pairs

The fluorescence induction and other fluorescence properties of spinach chloroplasts at room temperature were probed utilizing two 30-ps wide laser pulses (530 nm) spaced Δt (s) apart in time (Δt = 5–110 ns). The energy of the first pulse (P₁) was varied (10¹²–10¹⁶ photons · cm⁻²), while the energy of the second (probe) pulse (P₂) was held constant (5 · 10¹³ photons · cm⁻²). A gated (10 ns) optical multichannel analyzer-spectrograph system allowed for the detection of the fluorescence generated either by P₁ alone, or by P₂ alone (preceded by P₁). The dominant effect observed for the fluorescence yield generated by P₁ alone is the usual singlet-singlet exciton annihilation which gives rise to a decrease in the yield at high energies. However, when the fluorescence yield of dark-adapted chloroplasts is measured utilizing P₂ (preceded by pulse P₁) an increase in this yield is observed. The magnitude of this increase depends on Δt, and is characterized by a time constant of 28 ± 4 ns. This rise in the fluorescence yield is attributed to a reduction of the oxidized (by P₁) reaction center P-680⁺ by a primary donor. At high pulse energies (P₁ = 4 · 10¹⁴ photons · cm⁻²) the magnitude of this fluorescence induction is diminished by another quenching effect which is attributed to triplet excited states generated by intense P₁ pulses. Assuming that the P₁ pulse energy dependence of the fluorescence yield rise reflects the closing of the reaction centers, it is estimated that about 3–4 photon hits per reaction center are required to close completely the reaction centers, and that there are 185–210 chlorophyll molecules per Photosystem II reaction center.     

The electron-transfer kinetics of spinach ferredoxin with strong reductants

The reduction of spinach ferredoxin by V(II)-EDTA, Eu(II)-DPTA and propylene viologen was monitored electrochemically. The rates of these reactions were found to be 3.0 · 10⁴, 3.2 · 10⁵ and 1.2 · 10⁵ M⁻¹ · s⁻¹, respectively, by the use of chronoamperometry, pulse polarography, differential pulse polarography and rotating-disk voltammetry. These reaction rates were analyzed by tunneling theory for electron transfers, and the comparisons between the theoretical and experimental values were quite good. The tunneling theory also worked quite well with other ferredoxin electron-transfer rates that are available in the literature. In addition to that, the activation enthalpy and entropy compared well with the tunneling theory.     

Sulfide-induced oxygen uptake by isolated spinach chloroplasts catalyzed by photosynthetic electron transport

In addition to an inhibitory effect on the photoreduction of NADP⁺ by isolated spinach chloroplasts ( Spinacea oleracea L. cv. Melody Hybrid), sulfide initiated oxygen uptake by chloroplasts upon illumination, both in presence and absence of an electron acceptor. Sulfide-induced oxygen uptake was sensitive to DCMU demonstrating the involvement of photosynthetic electron transport. Addition of superoxide dismutase to the chloroplast suspension prevented the sulfide-induced oxygen uptake, which indicated that sulfide may be oxidized by the chloroplast, its oxidation being initiated by superoxide formed upon illumination (at the reducing side of PSI). Tris-induced inhibition of NADP⁺ photo-reduction could not be abolished by sulfide, which indicated that sulfide could not act as an electron donor for PSI.     

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.     

Thermoluminescence studies on photosynthetic energy conversion. II. Activation energies for three energy storage states associated with Photoreaction II of higher plants

Thermoluminescent glow curves of isolated chloroplasts were analyzed to determine activation energy, frequency factor and lifetime of the three glow peaks associated with Photoreaction II. Peak 1 had an activation energy of 0.8 eV, a frequency factor of 1.5 · 10¹⁴/s and a lifetime of 0.02 s. This glow peak appeared to be rather different from peaks 2 and 3, which were quite similar to each other. Peaks 2 and 3 gave activation energies respectively of 0.48 eV and 0.57 eV, frequency factors of 7 · 10⁷/s and 1 · 10⁹/s, and lifetimes of about 1.5 s. A second, less reliable method of analysis gave activation energies of 0.72 eV for peak 1 and 0.44 eV for peak 2. Our values are compared with those obtained by other workers, and the possible metastable states reflected by the three glow peaks are discussed.     

The mechanism of photosynthetic sulfate reduction by isolated chloroplasts

The reduction of sulfate by isolated spinach chloroplasts was studied. A reconstituted system of broken chloroplasts and of chloroplast extract reduced sulfate to sulfite in the light when ADP, NADP⁺, ferredoxin and glutathione were added. The chloroplast extract reduced sulfate to sulfite in the dark if supplemented with ATP and with reduced glutathione. Neither ferredoxin nor NADPH were needed for this reduction in the dark.

A sulfite reductase was purified from spinach leaves. Broken chloroplasts and sulfite reductase reduced sulfite to sulfide in the light when ferredoxin was added. NADP⁺ was not required for this reduction.

The results suggest that in chloroplasts a sulfate activated by ATP (phosphoadenosine phosphosulfate) is reduced to sulfite by a sulfhydryl compound and that sulfite is reduced to sulfide by a ferredoxin-dependent sulfite reductase.     

Effect of dicyclohexylcarbodiimide on the proton conductance of thylakoid membranes in intact chloroplast

The effects of dicyclohexylcarbodiimide, a potent inhibitor of chloroplast ATPase, on the light-induced electric potential changes in intact chloroplasts of Peperomia metallica and of a hornwort Anthoceros sp. were investigated by means of glass microcapillary electrodes. The characteristics of potential changes induced by flashes or continuous light in chloroplasts of both species are similar except for the phase of potential rise in continuous light, which is clearly biphasic in Anthoceros chloroplasts. Dicyclohexylcarbodiimide at concentration 5 · 10⁻⁵ M completely abolishes the transient potential undershoot in the light-off reaction but has little effect on the peak value of the photoelectric response. The membrane conductance in the light and in the dark was tested by measuring the decay kinetics of flash-generated potential in dark-adapted and preilluminated chloroplasts. In the absence of dicyclohexylcarbodiimide, preillumination causes a significant acceleration of the potential decay. The light-induced changes in the decay kinetics of flash-induced responses were abolished in the presence of dicyclohexylcarbodiimide, whereas the rate of potential decay in dark-adapted chloroplasts was not altered by dicyclohexylcarbodiimide. The results are consistent with the notion that dicyclohexylcarbodiimide diminishes H⁺ conductance of energized thylakoid membranes by interacting with the H⁺ channel of ATPase. The occurrence of a lag (approx. 300 ms) on the plot of potential undershoot (diffusion potential) versus illumination time might suggest the increase in H⁺ permeability coefficient of thylakoid membrane during illumination.     

Studies on the rate-limiting reaction of photosynthetic oxygen evolution in spinach chloroplasts

The modulated oxygen polarograph has been used to study the rate-determining steps of photosynthetic oxygen evolution in spinach chloroplasts. The rate constant, k, of the reaction has a value of 218±10 (S.E.) s⁻¹ at 23 °C and an activation energy of 7±2 (S.E.) kcal · mol⁻¹. A kinetic isotope experiment indicated that this step is probably not the water-splitting reaction. These findings resemble previous results with the unicellular alga Chlorella (Sinclair, J. and Arnason, T. (1974) Biochim. Biophys. Acta 368, 393–400). In other experiments we changed the pH, O₂ concentration and osmolarity of the medium, and treated the chloroplasts with 1 mM NH₄Cl without detecting any significant change in k. These results suggest that the step is irreversible. However, a significantly lower value of k, 110±20 (S.E.) s⁻¹ was obtained when all salts except 1 mM MgCl₂ were removed from the medium bathing the chloroplasts.     

The site of manganese function in photosynthetic electron transport system

The effects of Mn²⁺ on aerobic photobleaching of carotenoids, on photoreduction of 2,6-dichlorophenolindophenol (DCIP) and on fluorescence above 600 mμ of spinach chloroplasts washed with 0.8 M Tris-HC1 buffer were investigated. Carotenoids (mostly carotenes, lutein and violaxanthin) in the Tris-washed chloroplasts were irreversibly bleached by illumination with red light, while carotenoids in normal chloroplasts prepared with a low concentration of Tris-HC1 underwent no bleaching upon illumination. The photobleaching of carotenoids observed with Tris-washed chloroplasts was inhibited by Mn²⁺ (MnCl₂ or MnSO₄) as well as by some inhibitors of the Hill reaction such as dichlorophenyl-1,1-dimethylurea (DCMU), methylthio-4,6-bis-isopropylamino-s-triazine and o-phenanthroline or by reducing agents such as ascorbate plus tetramethyl-p-phenylene diamine (TMPD). DCIP photoreduction, which was deactivated by Tris, was reactivated to 50–80% of the rate for normal chloroplasts upon addition of Mn²⁺. The restored photoreduction of DCIP was inhibited by DCMU and carbonylcyanide m-chlorophenylhydrazone (CCCP). The steady-state fluorescence yield of normal chloroplasts measured at room temperature was lowered by Tris treatment, and the decreased yield was restored by adding Mn²⁺ as well as ascorbate plus TMPD. CCCP also lowered the yield; the yield was recovered by adding ascorbate plus TMPD. Determination of manganese in normal and Tris-washed chloroplasts showed that 30% of the manganese in chloroplast was removed with Tris. It was postulated that Mn²⁺ functions in the electron transport on the oxidizing side of Photosystem II at a site between water and an electron carrier (Y). CCCP as well as Tris inhibits the reduction of Y⁺ by Mn²⁺, and carotenoids are oxidized by Y⁺ which is reduced by ascorbate plus TMPD.     

The effect of temperature on the primary reaction of chloroplast Photosystem II Evidence for a temperature-dependent back reaction

The primary reaction of Photosystem II has been studied over the temperature range from −196 to −20 °C. The photooxidation of the reaction-center chlorophyll (P680) was followed by the free-radical electron paramagnetic resonance signal of P680⁺, and the photoreduction of the Photosystem II primary electron acceptor was monitored by the C-550 absorbance change.

At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680⁺ and the reduced acceptor.

The amount of reduced acceptor and P680⁺ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680⁺ is reduced by a secondary electron donor (cytochrome b₅₅₉) before P680⁺ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b₅₅₉ and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b₅₅₉ on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures.     

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Correlation of reaction-center chlorophyll (P-700) oxidation and bound iron-sulfur protein photoreduction in chloroplast Photosystem I at low temperatures

The extent of P-700 photooxidation at 18 °K has been followed in three different chloroplast preparations (unfractionated chloroplasts and two preparations enriched in Photosystem I). More than 90% of P-700⁺ formation in all preparations was eliminated by the addition of sodium dithionite at pH 10. Photoreduction of a bound chloroplast iron-sulfur protein was also decreased by at least 90% under similar conditions. Electron paramagnetic resonance spectra of the chloroplast preparations in the presence of dithionite showed chemical reduction of bound iron-sulfur protein under conditions where primary photochemistry is eliminated. These results indicate that P-700 photooxidation is concomitant with photoreduction of a bound iron-sulfur protein and that this iron-sulfur protein functions as the primary electron acceptor of Photosystem I.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Participation of Hydrogen Peroxide in the Inactivation of Calvin-Cycle SH Enzymes in SO2-Fumigated Spinach Leaves

In SO₂-fumigated spinach leaves under light, chloroplast SHenzymes, glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD)(EC 1.2.1.13 [EC] ), ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19 [EC] )and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11 [EC] ) weremore remarkably inactivated than other chloroplast enzymes.Their activities recovered after removal of SO₂. The inactivationparalleled light-dependent CO₂-fixation in spinach leaves. Inilluminated chloroplasts isolated from SO₂-fumigated spinachleaves, NADP-GAPD and Ru5PK were more specifically in activatedthan other chloroplast enzymes. These two enzymes could be protectedfrom the inactivation by adding catalase. The NADP-GAPD inactivationwas suppressed by DCMU, cytochrome c or anaerobic conditions.By adding thiol compounds, the NADP-GAPD inactivation was dischargedand the activity increased. In chloroplasts or crude extractsfrom non-fumigated spinach leaves, NADP-GAPD and Ru5PK weremore strongly inhibited by externally added H₂O₂ than otherchloroplast enzymes. All results supported the idea that thesuppression of photosynthesis at the beginning of SO₂ fumigationwas caused by the reversible inhibition of chloroplast SH enzymewith H₂O₂. (Received October 7, 1981; Accepted June 16, 1982)     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

The (Na + K)-dependent ATPase: mode of inhibition of ADP/ATP exchange activity by MgCl2

Na⁺-dependent ADP/ATP exchange activity, of a (Na⁺ + K⁺)-dependent ATPase preparation from eel electric organ, was measured in terms of the incorporation of ¹⁴C into ATP during incubations with unlabeled ATP and [¹⁴C]ADP. Estimates of initial rates of exchange were possible by keeping changes in nucleotide concentrations, from both exchange and extraneous hydrolytic processes, to less than 10%. Under these conditions, increases in MgCl₂ concentration, from 0.2 to 3 mM, generally inhibited this exchange activity. The concentrations of free Mg²⁺, Mg · ATP, and Mg · ADP present, with a range of MgCl₂, ATP, and ADP concentrations, were calculated from measured dissociation constants. Inhibition was associated with Mg · ATP as well as with Mg²⁺, at concentrations from 0.4 to 1 mM (Mg · ADP, in the same concentration range, probably inhibited also). The affinity of the enzyme for these inhibitors is in fair correspondence with demonstrated affinities for Mg²⁺, Mg · ATP, and Mg · ADP at low affinity substrate sites, measured kinetically. These observations are considered in terms of a dimeric enzyme with high and low affinity substrates sites: ADP/ATP exchange being catalyzed at the high affinity sites, with inhibition occurring through occupancy by Mg²⁺, Mg · ATP, or Mg · ADP, of the low affinity sites, thereby pulling the reaction process away from those steps involved in exchange.     

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.     

The dimerization of protochlorophyll pigments in non-polar solvents

The infrared, visible and nuclear magnetic resonance spectra of protochlorophyll a and vinylprotochlorophyll a in dry non-polar solvents (carbon tetrachloride, chloroform, cyclohexane) are presented and interpreted in terms of dimer interaction.

The infrared spectra in the 1600–1800 cm⁻¹ region clearly show the existence of a coordination interaction between the C-9 ketone oxygen function of one molecule and the central magnesium atom of another molecule. Infrared spectra in the OH stretching region (3200–3800 cm⁻¹) provide a valuable test of the water content in the samples.

The analysis of the absorption and circular dichroism spectra of protochlorophyll a and vinylprotochlorophyll a in carbon tetrachloride demonstrates the existence of a monomer-dimer equilibrium in the concentration range from 10⁻⁶ to 5 · 10⁻⁴ M. The dimerization constants are (6±2) · 10⁵ 1 · M⁻¹ for protochlorophyll a and (4.5±2) · 10⁵ 1 · M⁻¹ for vinylprotochlorophyll a at 20 °C. The deconvolution of visible spectra in the red region has been performed in order to obtain quantitative information on the dimer structure. Two models involving a parallel or a perpendicular arrangement of the associated molecules are considered.

From ¹H NMR spectra, it appears that the region of overlap occurs near ring V, in agreement with the interpretation of the infrared spectra.