Boris K. Semin

Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired photosystem II photochemical activity.

Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

Accumulation of Ferrous Iron in Chlamydomonas reinhardtii. Influence of CO2 and Anaerobic Induction of the Reversible Hydrogenase

The green alga, Chlamydomonas reinhardtii, can photoproduce molecular H2 via ferredoxin and the reversible [Fe]hydrogenase enzyme under anaerobic conditions. Recently, a novel approach for sustained H2 gas photoproduction was discovered in cell cultures subjected to S-deprived conditions (A. Melis, L. Zhang, M. Forestier, M.L. Ghirardi, M. Seibert [2000] Plant Physiol 122: 127–135). The close relationship between S and Fe in the H2-production process is of interest because Fe-S clusters are constituents of both ferredoxin and hydrogenase. In this study, we used Mössbauer spectroscopy to examine both the uptake of Fe by the alga at different CO2concentrations during growth and the influence of anaerobiosis on the accumulation of Fe. Algal cells grown in media with57Fe(III) at elevated (3%, v/v) CO2concentration exhibit elevated levels of Fe and have two comparable pools of the ion: (a) Fe(III) with Mössbauer parameters of quadrupole splitting = 0.65 mm s−1 and isomeric shift = 0.46 mm s−1 and (b) Fe(II) with quadrupole splitting = 3.1 mm s−1 and isomeric shift = 1.36 mm s−1. Disruption of the cells and use of the specific Fe chelator, bathophenanthroline, have demonstrated that the Fe(II) pool is located inside the cell. The amount of Fe(III) in the cells increases with the age of the algal culture, whereas the amount of Fe(II) remains constant on a chlorophyll basis. Growing the algae under atmospheric CO2 (limiting) conditions, compared with 3% (v/v) CO2, resulted in a decrease in the intracellular Fe(II) content by a factor of 3. IncubatingC. reinhardtii cells, grown at atmospheric CO2 for 3 h in the dark under anaerobic conditions, not only induced hydrogenase activity but also increased the Fe(II) content in the cells up to the saturation level observed in cells grown aerobically at high CO2. This result is novel and suggests a correlation between the amount of Fe(II) cations stored in the cells, the CO2 concentration, and anaerobiosis. A comparison of Fe-uptake results with a cyanobacterium, yeast, and algae suggests that the intracellular Fe(II) pool in C.reinhardtii may reside in the cell vacuole.

Effect of formate on Mössbauer parameters of the non-heme iron of PS II particles of cyanobacteria.

Mössbauer spectra were measured for PSII particles having an active water‐splitting system. The particles were isolated from the thennophilic cyanobacterium Synechococcus elongatus enriched in57Fe. The Mössbauer resonance absorption spectrum is a superposition of 3 doublets with the following quadrupole splitting and chemical shift: 1, δ = 0.40, Δ = 0.85; II, δ = 1.35,Δ =2.35; III, δ = 0.25, Δ = 1.65. The δ and Δ values of doublets I, II, III are characteristic of proteins with iron‐sulphur center, non‐heme iron of the reaction center of higher plants and of the oxidized cytochrome 6–559. Treatment with sodium formate to remove bicarbonate affects only the doublet of non‐heme iron, causing its quadrupole splitting to reduce to 1.75 and the chemical shift to reduce to 0.90. After washing out the formate, the Mossbauer spectrum of non‐heme iron is restored. The data suggest that bicarbonate is a ligand for the non‐heme iron of the reaction center of cyanobacteria.

Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca2+ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O2 evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as “the decoupling effect” (Semin et al. Photosynth Res 98:235–249, 2008). Extraction of Cl− from Ca2+-depleted membranes (PSII[–Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H2O2, and O2·−, as possible products of water oxidation in PSII(–Ca) membranes was examined. During the investigation of O2·− production in PSII(–Ca) samples, we found that (i) O2·− is formed on the acceptor side of PSII due to the reduction of O2; (ii) depletion of Cl− does not inhibit water oxidation, but (iii) Cl− depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl− under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O2, thereby increasing the rate of O2·− generation. From these observations we conclude that the product of water oxidation is H2O2 and that Cl− anions are not involved in the oxidation of water to H2O2 in decoupled PSII(–Ca) membranes. These results also indicate that Cl− anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H2O molecules to the Mn4CaO5 cluster and/or facilitate the release of H+ ions into the lumenal space.

Rapid Degradation of the Tetrameric Mn Cluster in Illuminated, PsbO-Depleted Photosystem II Preparations

A “decoupling effect” (light-induced electron transport without O2 evolution) was observed in Ca-depleted photosystem II (PSII(-Ca)) membranes, which lack PsbP and PsbQ (Semin et al. (2008) Photosynth. Res., 98, 235–249). Here PsbO-depleted PSII (PSII(-PsbO)) membranes (which also lack PsbP and PsbQ) were used to examine effects of PsbO on the decoupling. PSII(-PsbO) membranes do not reduce the acceptor 2,6-dichlorophenolindophenol (DCIP), in contrast to PSII(-Ca) membranes. To understand why DCIP reduction is lost, we studied light effects on the Mn content of PSII(-PsbO) samples and found that when they are first illuminated, Mn cations are rapidly released from the Mn cluster. Addition of an electron acceptor to PSII(-PsbO) samples accelerates the process. No effect of light was found on the Mn cluster in PSII(-Ca) membranes. Our results demonstrate that: (a) the oxidant, which directly oxidizes an as yet undefined substrate in PSII(-Ca) membranes, is the Mn cluster (not the YZ radical or P680+); (b) light causes rapid release of Mn cations from the Mn cluster in PSII(-PsbO) membranes, and the mechanism is discussed; and (c) rapid degradation of the Mn cluster under illumination is significant for understanding the lack of functional activity in some PSII(-PsbO) samples reported by others.

Mössbauer spectroscopy of iron metabolism and iron intracellular distribution in liver of rats

Mössbauer spectroscopy was used to investigate the distribution of iron in rat organs and its localisation in liver subcellular fraction. A 57Fe-sucrose complex solution was injected by 0.5 ml doses into tail veins of animals every day, during a 6-day period. Mössbauer spectra were measured in spleen, blood, liver and liver subcellular fractions. The mössbauer spectrum of a spleen sample has two symmetrical doublets, one with delta = 0.6 mm/s and delta = 0.7 mm/s, and the other with delta = 1.0 mm/s and delta = 2.35 mm/s. The Mössbauer spectrum of blood has parameters which are close to those for carboxyhemoglobin and oxyhemoglobin complexes. After the addition of sodium citrate, the proportion of the carboxyhemoglobin complex increases. The Mössbauer spectrum of liver has a two-component pattern with two symmetrical doublets, the first with delta = 0.6 mm/s and delta = 0.63 mm/s and the second with delta = 1.4 mm/s and delta = 3.45 mm/s. The first component, which was identified as ferritin, is present in all subcellular fractions (800 x gav sediment fraction, mitochondrial/lysosomal, microsomal and supernatant fractions), with its greatest content in microsomal fraction. After the addition of NaBH4 to mitochondrial/lysosomal fraction, about 20% of the iron contained in ferritin was reduced. In the Mössbauer spectrum this is reflected by an appearance of a doublet with delta = 0.85 mm/s and delta = 3.7 mm/s.

Investigation of the low-affinity oxidation site for exogenous electron donors in the Mn-depleted photosystem II complexes

In the manganese-depleted photosystem II (PSII[-Mn]) preparations, oxidation of exogenous electron donors is carried out through the high-affinity (HA) and the low-affinity (LA) sites. This paper investigates the LA oxidation site in the PSII(-Mn) preparations where the HA, Mn-binding site was blocked with ferric cations [[11] B.K. Semin, M.L. Ghirardi, M. Seibert, Blocking of electron donation by Mn(II) to Y(Z)(*) following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light, Biochemistry 41 (2002) 5854-5864.]. In blocked (PSII[-Mn,+Fe]) preparations electron donation by Mn(II) cations to Y(Z)(*) was not detected at Mn(II) concentration 10 microM (corresponds to K(m) for Mn(II) oxidation at the HA site), but detected at Mn concentration 100 microM (corresponds to K(m) for the LA site) by fluorescence measurements. Comparison of pH-dependencies of electron donation by Mn(II) through the HA and the LA sites revealed the similar pK(a) equal to 6.8. Comparison of K(m) for diphenylcarbazide (DPC) oxidation at the LA site and K(d) for A(T) thermoluminescence band suppression by DPC in PSII(-Mn,+Fe) samples suggests that there is relationship between the LA site and A(T) band formation. The role of D1-His190 as an oxidant of exogenous electron donors at the LA site is discussed. In contrast to electrogenic electron transfer from Mn(II) at the HA site to Y(Z)(*), photovoltage due to Mn(II) oxidation in iron-blocked PSII(-Mn) core particles was not detected.

Coordination sphere and structure of the Mn cluster of the oxygen-evolving complex in photosynthetic organisms

The great similarity between the binding of Fe(II) and the high‐affinity Mn‐binding site in the Mn‐depleted PSII membranes (Semin et al. (1996) FEBS Lett. 375, 223–226) suggests that the coordination sphere of Mn in PSII is also suitable for iron. A comparison is performed between the primary amino acid sequences of D1 and D2 and diiron‐oxo enzymes with the function of oxygen activation. All conservative motifs (EXXH) and residues binding and stabilizing the diiron cluster in diiron‐oxo enzymes have been found in the C‐terminal domains of D1 and D2 polypeptides. On the basis of these sequence similarities we suggest a structural model for the manganese cluster in the oxygen‐evolving complex.

High-specific binding of Fe(II) at the Mn-binding site in Mn-depleted PSII membranes from spinach

The interaction of Fe(II) and Fe(III) with the ‘high‐affinity Mn‐binding site’ in Mn‐depleted photosystem II (PSII) was investigated using diphenilcarbazide (DPC)/2, 6‐dichlorophenol‐indophenol (DCIP) inhibition assay. Fe(III) was ineffective in the inhibition of DPC‐DCIP reaction while Fe(II) decreased the rate of DCIP photoreduction supported by DPC in the same concentration range as Mn(II). The effectivity of the interaction of Fe(II) with the high affinity Mn‐binding site depends on different anions in the same manner as for Mn(II) and coincides with hierarchy observed for the stimulation of O2 evolution. The Fe(II) binding is accompanied by its oxidation. By using reductants it was shown that the high affinity site contains a redox‐active component and the reduction of this component totally prevents the binding of Fe(II).

Photosystem II of peas: effects of added divalent cations of Mn, Fe, Mg, and Ca on two kinetic components of P-680(+) reduction in Mn-depleted core particles

The catalytic Mn cluster of the photosynthetic oxygen-evolving system is oxidized via a tyrosine, Y(Z), by a photooxidized chlorophyll a moiety, P(+)(680). The rapid reduction of P(+)(680) by Y(Z) in nanoseconds requires the intactness of an acid/base cluster around Y(Z) with an apparent functional pK of <5. The removal of Mn (together with bound Ca) shifts the pK of the acid/base cluster from the acid into the neutral pH range. At alkaline pH the electron transfer (ET) from Y(Z) to P(+)(680) is still rapid (<1 micros), whereas at acid pH the ET is much slower (10-100 micros) and steered by proton release. In the intermediate pH domain one observes a mix of these kinetic components (see R. Ahlbrink, M. Haumann, D. Cherepanov, O. Bögershausen, A. Mulkidjanian, W. Junge, Biochemistry 37 (1998)). The overall kinetics of P(680)(+) reduction by Y(Z) in Mn-depleted photosystem II (PS II) has been previously shown to be slowed down by divalent cations (added at >10 microM), namely: Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+) (C.W. Hoganson, P.A. Casey, O. Hansson, Biochim. Biophys. Acta 1057 (1991)). Using Mn-depleted PS II core particles from pea as starting material, we re-investigated this phenomenon at nanosecond resolution, aiming at the effect of divalent cations on the particular kinetic components of P(+)(680) reduction. To our surprise we found only the slower, proton steered component retarded by some added cations (namely Co(2+)/Zn(2+)>Fe(2+)>Mn(2+)). Neither the fast component nor the apparent pK of the acid/base cluster around Y(Z) was affected. Apparently, the divalent cations acted (electrostatically) on the proton release channel that connects the oxygen-evolving complex with the bulk water, but not on the ET between Y(Z) and P(+)(680), proper. Contrastingly, Ca(2+) and Mg(2+), when added at >5 mM, accelerated the slow component of P(+)(680) reduction by Y(Z) and shifted the apparent pK of Y(Z) from 7.4 to 6.6 and 6.7, respectively. It was evident that the binding site(s) for added Ca(2+) and Mg(2+) were close to Y(Z) proper. The data obtained are discussed in relation to the nature of the metal-binding sites in photosystem II.