Vyacheslav V. Klimov

The origin of atmospheric oxygen on Earth: The innovation of oxygenic photosynthesis

The evolution of O2-producing cyanobacteria that use water as terminal reductant transformed Earths atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO2) is a more efficient alternative substrate than water for O2 production by oxygenic phototrophs. This analysis clarifies the origin of the long debated “bicarbonate effect” on photosynthetic O2 production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O2 evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.

Three types of Photosystem II photoinactivation : I. Damaging processes on the acceptor side.

Oxygen evolving photosystem II particles were exposed to 100 and 250 W m−2 white light at 20°C under aerobic, anaerobic and strongly reducing (presence of dithionite) conditions. Three types of photoinactivation processes with different kinetics could be distinguished: (1) The fast process which occurs under strongly reducing (t1/2≅1–3 min) and anaerobic conditions (t1/2≅4–12 min). (2) The slow process (t1/2≅15–40 min) and (3) the very slow process (t1/2>100 min), both of which occur under all three sets of conditions.The fast process results in a parallel decline of variable fluorescence (Fv) and of Hill reaction rate, accompanied by an antiparallel increase of constant fluorescence (Fo). We assume that trapping of QA in a negatively charged stable state, (QA−)stab, is responsible for the effects observed.The slow process is characterized by a decline of maximal fluorescence (Fm). In presence of oxygen this decline is due to the well known disappearance of Fv which proceeds in parallel with the inhibition of the Hill reaction; Fo remains essentially constant. Under anaerobic and reducing conditions the decline of Fm represents the disappearance of the increment in Fo generated by the fast process. We assume that the slow process consists in neutralization of the negative charge in the domain of QA in a manner that renders QA non-functional. The charge separation in the RC is still possible, but energy of excitation becomes thermally dissipated.The very slow photoinactivation process is linked to loss of charge separation ability of the PS II RC and will be analyzed in a forthcoming paper.

EFFECT OF EXTRACTION AND RE-ADDITION OF MANGANESE ON LIGHT REACTIONS OF PHOTOSYSTEM-II PREPARATIONS

Manganese plays an important role in photosynthetic oxidation of H20 (reviews [l-3]). Reaction centers (RC) of photosystem II (PS II) carry out successive 4-step oxidation of a special (Mncontaining) enzymatic system which in turn oxidizes Hz0 [l-3]. The minimal quantity of Mn necessary for 02 evolution is 5-6 atoms/400 chlorophyll (chl) molecules or /l RC of PS II [l-4]. The greater part (-2/3rds) of this Mn is ‘loosely bound’ and can be easily extracted by alkaline Tris, NH20H, Triton X-100 or by heating, and the extraction leads to inhibition of 02 evolution and associated light reactions of PS II [l-lo]. ‘Firmly bound’ Mn ( 1/3rd of the pool) which remains in PS II after the extraction procedures seems not to be required for electron transport in PS II 141. However, up to 70% of Mn can be removed from chloroplasts without essential loss of their ability to evolve 02 [ll]. Reported characteristics of EPR spectra of Mn in chloroplasts [ 12151 may indicate participation of either 4 or 2 atoms of Mn in PS II reactions. of Mg2+ or any other divalent cation of metals, M2+). New results from a thorough investigation of these effects reported here show that activity of the Mn-containing system in the donor side of PS II requires 4 Mn atoms, 2 of which can be replaced by either Mg2+ or some other divalent metal ions (M2+).

The photoproduction of superoxide radicals and the superoxide dismutase activity of Photosystem II. The possible involvement of cytochrome b559

In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl2-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c1/2=10 μM TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c1/2≈ 3 μM. 3.) The photoproduction of O2− is coupled with H+-uptake. This effect is diminished by the addition of the O2−-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O2− in the light if a PS II electron donor is added.Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.

Glycinebetaine protects the D1/D2/Cytb559 complex of photosystem II against photo-induced and heat-induced inactivation

The presence of 1.0 mol/L glycinebetaine during isolation of D1/D2/Cytb559 reaction centre (RC) complexes from photosystem II (PSII) membrane fragments preserved the photochemical activity, monitored as the light-induced reduction of pheophytin and electron transport from diphenylcarbazide to 2.6-dichlorophenol-indophenol.-Glycinebetaine also protected the D1/D2/Cytb559 complexes against strong light-induced damage to the photochemical reactions and the irreversible bleaching of beta-carotene and chlorophyll. The presence of glycinebetaine also enhanced thermotolerance of the D1/D2/Cytb559 complexes isolated in the presence of 1.0 mol/L betaine with an increase in the temperature for 50% inactivation from 29 degrees C to 35 degrees C. The results indicate an increased supramolecular structural stability in the presence of glycinebetaine.

A photosystem II‐associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution

We show for the first time that Cah3, a carbonic anhydrase associated with the photosystem II (PSII) donor side in Chlamydomonas reinhardtii, regulates the water oxidation reaction. The mutant cia3, lacking Cah3 activity, has an impaired water splitting capacity, as shown for intact cells, thylakoids and PSII particles. To compensate this impairment, the mutant overproduces PSII reaction centres (1.6 times more than wild type). We present compelling evidence that the mutant has an average of two manganese atoms per PSII reaction centre. When bicarbonate is added to mutant thylakoids or PSII particles, the O2 evolution rates exceed those of the wild type by up to 50%. The donor side of PSII in the mutant also exhibits a much higher sensitivity to overexcitation than that of the wild type. We therefore conclude that Cah3 activity is necessary to stabilize the manganese cluster and maintain the water‐oxidizing complex in a functionally active state. The possibility that two manganese atoms are enough for water oxidation if bicarbonate ions are available is discussed.

In photoinhibited photosystem II particles pheophytin photoreduction remains unimpaired

Oxygen‐evolving photosystem II particles (DT 20) isolated from pea chloroplasts by digitonin‐Triton X‐100 fractionation were photoinhibited with 150 W·m−2 white light, at 20°C under three conditions: aerobic, anaerobic and strongly reducing (E h poised to approx. −250 mV with dithionite). Hill reaction rate (H2O → BQ) and variable fluorescence (F v) declined in parallel in all three cases with shortening half times: 30, 10 and 2.5 min, respectively. Light‐induced absorbance changes at 685 nm characteristic of reversible photoaccumulation of reduced pheophytin (E h ≈ −250 mV) remained essentially unchanged. We conclude that the three types of photoinhibitory treatment do not impair the separation of charges between chlorophyll P‐680 and pheophytin in the photosystem II reaction center.

Bicarbonate requirement for the donor side of photosystem II

Suppression of electron flow (and its subsequent restoration with 3–10 mM NaHCO3) on the donor side of photosystem II is shown upon either a partial depletion of pea subchloroplast membranes in bicarbonate or the addition of 5–20 μM formate. At higher concentrations (5 mM) formate induces the known ‘bicarbonate effect’ on the acceptor side of photosystem II. In preparations depleted of manganese the restoration of electron flow with 0.1–0.2 μM MnCl2 (2–4 Mn per photosystem II reaction center) occurs only in the presence of bicarbonate and it is accompanied by an increased functional binding of manganese. Restoration of electron flow with diphenylcarbazide or NH2OH does not require the addition of NaHCO3. It is suggested that bicarbonate participates in the formation of the Mn‐cluster capable of water oxidation or serves as a substrate for the water‐oxidizing center.

Stabilization of oxygen evolution and primary electron transport reactions in photosystem II against heat stress with glycinebetaine and sucrose

The protective action of co-solutes, such as sucrose and glycinebetaine, against the thermal inactivation of photosystem II function was studied in untreated and Mn-depleted photosystem II preparations. It was shown that, in addition to the reactions that depend on the oxygen evolving activity of the photosystem, those that implicate more intimately the reaction center itself are protected by high concentrations of osmolytes. However, the temperature required to inhibit oxygen evolution totally in the presence of osmolytes is lower than that required to eliminate reactions, such as P680 (primary electron donor in photosystem II) photo-oxidation and pheophytin photo reduetion, which only involve charge separation and primary electron transport processes. The energy storage measured from the thermal dissipation yield during photoacoustic experiments and the yield of variable fluorescence are also protected to a significant degree (up to 30%) at temperatures at which oxygen evolution is totally inhibited. It is suggested that a cyclic electron transport reaction around photosystem II may be preserved under these conditions and may be responsible for the energy storage measured at relatively high temperatures. This interpretation is also supported by thermoluminescence data involving the recombination between reduced electron acceptors and oxidized electron donors at - 30 and - 55 °C. The data also imply that a high concentration of osmolyte allows the stabilization of the photosystem core complex together with the oxygen-evolving complex. The stabilization effect is understood in terms of the minimization of protein-water interactions as proposed by the theory of Arakawa and Timasheff (Biophys. J., 47 (1985) 411--414).

Bicarbonate requirement for the water-oxidizing complex of photosystem II

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.