The reduction kinetics of chlorophyll a1 as an indicator for proton uptake between the light reactions in chloroplasts

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll a₁ (P-700) after far-red preilluination have been studied with high time resolution in spinach chloroplasts.

1. 1. The kinetics of chlorophyll a₁ exhibits a pronounced lag phase of 2–3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II.

2. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction.

3. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values.

4. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll a₁. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules.

5. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylakoid membrane.

6. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll a₁ reduction, is estimated as > 10¹¹ M⁻¹ · s⁻¹. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution.

Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

Electron transport between plastoquinone and chlorophyll aI in chloroplasts

After preillumination with System I light spinach chloroplasts were excited by one flash or a group of saturating flashes. During the following dark period the time courses of the oxidation of plastohydroquinone and of the simultaneous reduction of oxidized cytochrome f and chlorophyll a_I (P700) have been measured.

1. 1. From a correlation of these kinetics it can be concluded that at least 85% of the electrons from plastohydroquinone are transferred to chlorophyll a_I.

2. 2. After one flash 93% of the oxidized chlorophyll a_I is reduced. This suggests a high equilibrium constant between chlorophyll a_I and its donor as well as an equilibration between different chlorophyll a_I molecules.

3. 3. Cytochrome f is also reduced by plastohydroquinone. A ratio of active cytochrome f to chlorophyll a_I of 0.4:1 is observed. The half-life time of the reduction of cytochrome f is 17 ms. The time course indicates that in the dark cytochrome f does not transfer electrons to chlorophyll a_I and that no more than 15% of the electron transport passes cytochrome f. Therefore cytochrome f should be situated in a side path of the linear electron transport.

4. 4. The electrons which are released from plastohydroquinone and are not accepted by oxidized cytochrome f and chlorophyll a_I have been calculated. From this difference properties of an electron carrier, as yet not identified, between plastoquinone and chlorophyll a_I are predicted.

Mechanistic features of lignin peroxidase-catalyzed oxidation of substituted phenols and 1,2-dimethoxyarenes

The steady state kinetic parameters Km and kcat for the oxidation of phenolic substrates by lignin peroxidase correlated with the presteady state kinetic parameters Kd and k for the reaction of the enzyme intermediate compound II with the substrates, indicating that the latter is the rate-limiting step in the catalytic cycle. ln Km and ln Kd values for phenolic substrates correlated with redox properties, unlike ln kcat and ln k. This finding suggests that in contrast to horseradish peroxidase, electron transfer is not the rate-limiting step during oxidation by lignin peroxidase compound II. A mechanism is proposed for lignin peroxidase compound II reactions consisting of an equilibrium electron transfer step followed by a subsequent rate-limiting step. Analysis of the correlation coefficients for linear relationships between ln Kd and ln Km and different calculated redox parameters supports a mechanism in which the acidic forms of phenols are oxidized by lignin peroxidase and electron transfer is coupled with proton transfer. 1,2-Dimethoxyarenes did not comply with the trend for phenolic substrates, which may be a result of more than one substrate binding site on lignin peroxidase and/or alternative binding modes. This behavior was supported by analogue studies with the 1,2-dimethoxyarenes veratric acid and veratryl aldehyde, both of which are not oxidized by lignin peroxidase. Inclusion of either had little effect on the rate of oxidation of phenolic substrates yet resulted in a decrease in the oxidation rate of 1,2-dimethoxyarene substrates, which was considerable for veratryl alcohol and less pronounced for 3,4-dimethoxyphenethylalcohol and 3,4-dimethoxycinnamic acid, in particular in the presence of veratric acid.     

Effects of far-red light on fluorescence induction in infiltrated pea leaves under diminished ΔpH and Δφ components of the proton motive force

Chlorophyll fluorescence induction curves induced by an actinic pulse of red light follow different kinetics in dark-adapted plant leaves and leaves preilluminated with far-red light. This influence of far-red light was abolished in leaves infiltrated with valinomycin known to eliminate the electrical (Δφ) component of the proton-motive force and was strongly enhanced in leaves infiltrated with nigericin that abolishes the ΔpH component. The supposed influence of ionophores on different components of the proton motive force was supported by differential effects of these ionophores on the induction curves of the millisecond component of chlorophyll delayed fluorescence. Comparison of fluorescence induction curves with the kinetics of P700 oxidation in the absence and presence of ionophores suggests that valinomycin facilitates a build-up of a rate-limiting step for electron transport at the site of plastoquinone oxidation, whereas nigericin effectively removes limitations at this site. Far-red light was found to be a particularly effective modulator of electron flows in chloroplasts in the absence of ΔpH backpressure on operation of the electron-transport chain.     

The ratio of the two light reactions and their coupling in chloroplasts.

The kinetics of the absorbance changes of chlorophyll alphaI (P-700) and plastoquinone induced by xenon flashes of saturating intensity were studied in spinach chloroplasts. 1. The total amount of chlorophyll alphaI is compared with that amount being reduced via the rate-limiting step between the light reactions. This is based on the amplitudes of the absorbance changes of chlorophyll alphaI after chemical reduction and after a group of flashes following far-red preillumination. It is concluded that only 75% of chlorophyll alphaI is coupled to chlorophyll alphaII via linear electron transport and that the remaining 25% is functionally isolated. 2. A ratio of 0.85 for coupled chlorophyll alphaI to chlorophyll alphaII is estimated from the time course of the absorbance changes of plastoquinone and chlorophyll alphaI in two independent ways. 3. The oxygen yield per flash is used to calculate the difference extinction coefficient of chlorophyll alphaI at the maximum of the red absorbance band in spinach chloroplasts: delta xi703 = (6.7 +/- 0.7)-10(4) M-1-cm-1. The assumption of a quantitative electron transfer from water via plastoquinone to coupled chlorophyll alphaI is supported by the same extinction coefficient reported by Hiyama and Ke for Photosystem I particles. The location and function of the different chlorophylls alphaI is discussed in detail.     

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.     

Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase

This review describes the development and application of photoactive ruthenium complexes to study electron transfer and proton pumping reactions in cytochrome c oxidase (CcO). CcO uses four electrons from Cc to reduce O(2) to two waters, and pumps four protons across the membrane. The electron transfer reactions in cytochrome oxidase are very rapid, and cannot be resolved by stopped-flow mixing techniques. Methods have been developed to covalently attach a photoactive tris(bipyridine)ruthenium group [Ru(II)] to Cc to form Ru-39-Cc. Photoexcitation of Ru(II) to the excited state Ru(II*), a strong reductant, leads to rapid electron transfer to the ferric heme group in Cc, followed by electron transfer to Cu(A) in CcO with a rate constant of 60,000s(-1). Ruthenium kinetics and mutagenesis studies have been used to define the domain for the interaction between Cc and CcO. New ruthenium dimers have also been developed to rapidly inject electrons into Cu(A) of CcO with yields as high as 60%, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism.     

Water oxidation by photosystem II: H(2)O-D(2)O exchange and the influence of pH support formation of an intermediate by removal of a proton before dioxygen creation

Understanding the chemistry of photosynthetic water oxidation requires deeper insight into the interrelation between electron transfer (ET) and proton relocations. In photosystem II membrane particles, the redox transitions of the water-oxidizing Mn complex were initiated by nanosecond laser flashes and monitored by absorption spectroscopy at 360 nm (A(360)). In the oxygen evolution transition (S(3) + hν → S(0) + O(2)), an exponential decrease in A(360) (τ(O(2)) = 1.6 ms) can be assigned to Mn reduction and O(2) formation. The corresponding rate-determining step is the ET from the Mn complex to a tyrosine radical (Y(Z)(ox)). We find that this A(360) decrease is preceded by a lag phase with a duration of 170 ± 40 μs (τ(lag) at pH 6.2), indicating formation of an intermediate before ET and O-O bond formation and corroborating results obtained by time-resolved X-ray spectroscopy. Whereas τ(O(2)) exhibits a minor kinetic isotope effect and negligible pH dependence, formation of the intermediate is slowed significantly both in D(2)O (τ(lag) increase of ～140% in D(2)O) and at low pH (τ(lag) of 30 ± 20 μs at pH 7.0 vs τ(lag) of 470 ± 80 μs at pH 5.5). These findings support the fact that in the oxygen evolution transition an intermediate is created by deprotonation and removal of a proton from the Mn complex, after Y(Z)(ox) formation but before the onset of electron transfer and O-O bond formation.     

Thylakoids from pea seedlings grown under intermittent light: biochemical and flash-spectrophotometric properties.

Thylakoid membranes were isolated from pea seedlings grown under intermittent light (2-min light/118-min dark cycles). These preparations differed from controls (thylakoids from plants grown under 16-h light/8-h dark cycles) in the following respects: 15 times smaller chlorophyll/protein ratio, 10 times greater chlorophyll a/b ratio, absence of light-harvesting chlorophyll a/b binding proteins, and 2-3-fold greater ratio of photosystem II over photosystem I. In addition we found the following: (1) Electrogenic electron transfer around cytochrome b6/f under flashing light was greatly enhanced, probably as a consequence of the greater photosystem II/photosystem I ratio. (2) The rate of proton uptake from the medium at the acceptor side of photosystem II was enhanced, probably by unshielding of the quinone binding domain. (3) The N,N'-dicyclohexylcarbodiimide sensitivity of the proton-pumping activity of photosystem II was absent, which was consistent with the attribution of a N,N'-dicyclohexylcarbodiimide-induced protonic short circuit to chlorophyll a/b binding proteins. (4) The sensitivity of oxygen evolution under continuous light to variations of pH or the concentration of Ca2+ was altered. Chlorophyll a/b binding proteins serve as light-harvesting antennas. We found in addition that they modulated the activity of water oxidation and, in particular, the proteolytic reactions around photosystem II.     

Effect of pH on the oxidase activity of ceruloplasmin

The effect of pH on the kinetic parameters (Kms, Vs) of the reaction of adrenaline and Fe(II) (More's salt) oxidation by ceruloplasmin isolated from human donor blood was investigated. It was assumed that the imidazole group of histidine is functionally important for the above reactions. For Fe(II) the effect of the ionizeable group was observed during substrate binding to the ceruloplasmin molecule, whereas in the course of the adrenaline oxidation reaction it manifests itself during catalytic interaction of the substrate with the enzyme. The organic substrate can bind both to the protonated and to the non-protonated form of the enzyme. Fe(II) interacts only with the protonated form of the protein. In both cases, the rate-limiting step of the oxidase reaction is preceded by a single step, i.e., proton binding. The schemes describing the order of proton attachment in the course of the above reactions are proposed.     

The photosynthetic water oxidase: its proton pumping activity is short-circuited within the protein by DCCD

The photosynthetic water oxidase is composed of ˜15 polypeptides which are grouped around two functional parts: photosystem II and the catalytic manganese centre. Photochemically driven vectorial electron transfer between the manganese centre and bound plastoquinone causes deprotonation–protonation reactions at opposite sides of the thylakoid membrane. Thereby the water oxidase acts as a proton pump. Incubation of stacked thylakoids with N,N'-dicyclohexylcarbodiimide (DCCD) short-circuited its proton pumping activity. Under flashing light, the extent of both proton release into the lumen by water oxidation and of proton uptake from the medium by reduced quinone was diminished. Instead there was a rapid electrogenic backreaction with a strong H/D-isotope effect. Apparently protons which were produced by water oxidation were channelled across the transmembrane protein to the bound quinone. A more rapid protonation of the reduced quinone was evident from a shortening of the time lag for the reduction of photosystem I. These effects were paralleled by the preferential labelling with [¹⁴C]DCCD in stacked thylakoids of two polypeptides with 20 and 24 kd apparent molecular mass. These may be capping the oxidizing and the reducing terminus of the water oxidase to control proton extrusion and proton uptake respectively.     

The rate-limiting step and nonhyperbolic kinetics in the oxidation of ferrocytochrome c catalyzed by cytochrome c oxidase

The level of reduction of cytochrome a and CuA during the oxidation of ferrocytochrome c has been determined in stopped-flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, CuA is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and CuA to cytochrome a3-CuB is a two-electron process and, in addition, that the binding of oxidized cytochrome c to the electron- transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate-limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps.     

Water, water everywhere, and its remarkable chemistry

Photosystem II (PSII), the multisubunit pigment-protein complex localised in the thylakoid membranes of oxygenic photosynthetic organisms, uses light energy to drive a series of remarkable reactions leading to the oxidation of water. The products of this oxidation are dioxygen, which is released to the atmosphere, and reducing equivalents destined to reduce carbon dioxide to organic molecules. The water oxidation occurs at catalytic sites composed of four manganese atoms (Mn(4)-cluster) and powered by the redox potential of an oxidised chlorophyll a molecule (P680(*+)). Gerald T (Jerry) Babcock and colleagues showed that electron/proton transfer processes from substrate water to P680(*+) involved a tyrosine residue (Y(Z)) and proposed an attractive reaction mechanism for the direct involvement of Y(Z) in the chemistry of water oxidation. The 'hydrogen-atom abstract/metalloradical' mechanism he formulated is an expression of his genius and a highlight of his many other outstanding contributions to photosynthesis research. A structural basis for Jerry's model is now being revealed by X-ray crystallography.     

Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.

The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.     

Reaction kinetics of some important site-specific endonucleases.

Reaction kinetics of the site-specific endonucleases BamHI, BgIII, C1aI, EcoRI, HpaII, PstI, SaII, SmaI, and XorII were investigated employing some frequently used substrates. Six of these enzymes could be analyzed under steady-state conditions. Kinetic data were obtained from progress curves applying an integrated Michaelis-Menten equation. KM ranged from 4 x 10(-9) M to 4 x 10(-11) M. Activities also spanned two orders of magnitude. In the case of C1aI the analysis of the pre-steady-state kinetics ("burst reaction") allowed the assessment of several rate constants. The rate-limiting step is the very slow dissociation of the enzyme-product complex (0.22 min(-1)). This complex is formed from the enzyme-bound nicked intermediate at a rate of 1.7 min(-1). The introduction of the first cut is again faster by a factor of about 6. SmaI and XorII resembled C1aI in their kinetics. The burst reaction can be used for the easy and unambiguous determination of molar concentrations of site-specific endonucleases in any preparation, which is free of non-specific DNases.     

Proton release during the redox cycle of the water oxidase

Old and very recent experiments on the extent and the rate of proton release during the four reaction steps of photosynthetic water oxidation are reviewed. Proton release is discussed in terms of three main sources, namely the chemical production upon electron abstraction from water, protolytic reactions of Mn-ligands (e.g. oxo-bridges), and electrostatic response of neighboring amino acids. The extent of proton release differs between the four oxidation steps and greatly varies as a function of pH both, but differently, in thylakoids and PS II-membranes. Contrastingly, it is about constant in PS II-core particles. In any preparation, and on most if not all reaction steps, a large portion of proton transfer can occur very rapidly (<20 s) and before the oxidation of the Mn-cluster by Y_z ⁺ is completed. By these electrostatically driven reactions the catalytic center accumulates bases. An additional slow phase is observed during the oxygen evolving step, S₃S₄S₀. Depending on pH, this phase consists of a release or an uptake of protons which accounts for the balance between the number of preformed bases and the four chemically produced protons. These data are compatible with the hypothesis of concerted electron/proton-transfer to overcome the kinetic and energetic constraints of water oxidation.Abbreviations BBY-membranes Photosystem II-enriched membrane fragments prepared after Berthold, Babcock and Yocum (1981) - BSA bovine serum albumin - Chl chlorophyll - CAB-protein chlorophyll a/b-binding protein - core particles oxygen evolving reaction center core particles of Photosystem II - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - IML intermittent light - P-680 primary electron donor of Photosystem II - PS II Photosystem II - Y_z tyrosine residue on the D1 polypeptide, electron carrier between manganese and P-680 - photochemical reaction      

Electron-Transfer-Induced Acidity/Basicity and Reactivity Changes of Purine and Pyrimidine Bases. Consequences of Redox Processes for Dna Base Pairs

Changes in the oxidation state of the DNA bases, induced by oxidation (ionization) or by reduction (electron capture), have drastic effects on the acidity or basicity, respectively, of the molecules. Since in DNA every base is connected to its complementary base in the other strand, any change of the electric charge status of a base in one DNA strand that accompanies its oxidation or reduction may affect also the other strand via proton transfer across the hydrogen bonds in the base pairs. The free energies for electron transfer to or from a base can be drastically altered by the proton transfer processes that accompany the electron transfer reactions. Electron-transfer (ET) induced proton transfer sensitizes the base opposite to the ET-damaged base to redox damage, i.e., damage produced by separation of charge (ionization) has an increased change of being trapped in a base pair. Of the two types of base pair in DNA, A-T and C-G, the latter is more sensitive to both oxidative and reductive processes than the former.

Proton transfer induced by ET does not only occur between the heteroatoms (o and N) of the base pairs (intra-pair proton transfer), but also to and from adjacent water molecules in the hydration shell of DNA (extra-pair proton transfer). These proton transfers can involve carbon and as such are likely to be irreversible. It is the A-T pair which appears to be particularly prone to such irreversible reactions.     

Dissecting the pattern of proton release from partial process involved in ubihydroquinone oxidation in the Q-cycle

A key feature of the modified Q-cycle of the cytochrome bc₁ and related complexes is a bifurcation of QH₂ oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides, using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H⁺are released on complete turnover of the Q-cycle (2H⁺/ubiquinol (QH₂) oxidized). We identified different partial processes of the Q_o-site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron?sulfur protein. In the absence of Q_o-site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q_o-site, involving formation of the ES-complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH₂ on both turnovers. However, a minor lag in proton release found at pH?7 but not at pH?8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a pK in that range.     

Is there a rate-limiting step in the catalytic cycle of [Ni-Fe] hydrogenases?

The question of the existence of a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases was taken up by using the sets of data available in the case of two specific enzymes: the hydrogenase from Thiocapsa roseopercisina, in which isotope effects have been systematically investigated over a wide pH range, and the enzyme from Desulfovibrio fructosovorans, for which the activities and the redox properties have been studied in two different forms, the wild type and the P238C mutant. When these data are analyzed in the light of appropriate kinetic models, it is concluded that electron transfer and proton transfer are rate limiting in the H2 uptake and H2 evolution reactions, respectively. This proposal is consistent with the data available from other Ni-Fe enzymes.