Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Study of photosystem 2 heterogeneity in the sulfur-deficient green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

A set of chlorophyll fluorescence methods, including PEA (Plant Efficiency Analyser), PAM (Pulse Amplitude Modulated fluorometer), and picosecond fluorometer, was employed to study PS 2 heterogeneity in sulfur deprived green algae Chlamydomonas reinhardtii. The regression method and JIP test were applied to analyze chlorophyll fluorescence kinetics. The fractions of PS 2 characterized by the energetic disconnection, smaller antenna size, elevated constant rate of primary photochemistry, and inability to maintain ΔpH-dependent energy dissipation increased essentially already after 12 h of incubation in sulfur depleted medium. The amount of PS 2 centers with reduced Q_A (closed state), Q_B-non-reducing centers with impaired water splitting function, and centers coupled to the plastoquinone pool with the slow cycle rate increased dramatically after 24 h period of deprivation. The mechanisms of PS 2 inactivation under sulfur deprivation are discussed.     

Dynamics of photosystem II heterogeneity in Dunaliella salina (green algae)

Based on the electron-transport properties on the reducing side of the reaction center, photosystem II (PS II) in green plants and algae occurs in two distinct forms. Centers with efficient electron-transport from Q_A to plastoquinone (Q_B-reducing) account for 75% of the total PS II in the thylakoid membrane. Centers that are photochemically competent but unable to transfer electrons from Q_A to Q_B (Q_B-nonreducing) account for the remaining 25% of total PS II and do not participate in plastoquinone reduction. In Dunaliella salina, the pool size of Q_B-nonreducing centers changes transiently when the light regime is perturbed during cell growth. In cells grown under moderate illumination intensity (500 E m^-2s^-1), dark incubation induces an increase (half-time 45 min) in the Q_B-nonreducing pool size from 25% to 35% of the total PS II. Subsequent illumination of these cells restores the steady-state concentration of Q_B-nonreducing centers to 25%. In cells grown under low illumination intensity (30 µE m^–2s^–1), dark incubation elicits no change in the relative concentration of Q_B-nonreducing centers. However, a transfer of low-light grown cells to moderate light induces a rapid (half-time 10 min) decrease in the Q_B-nonreducing pool size and a concomitant increase in the Q_B-reducing pool size. These and other results are explained in terms of a pool of Q_B-nonreducing centers existing in a steady-state relationship with Q_B-reducing centers and with a photochemically silent form of PS II in the thylakoid membrane of D. salina. It is proposed that Q_B-nonreducing centers are an intermediate stage in the process of damage and repair of PS II. It is further proposed that cells regulate the inflow and outflow of centers from the Q_B-nonreducing pool to maintain a constant pool size of Q_B-nonreducing centers in the thylakoid membrane.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F_o non-variable fluorescence yield - F_pl intermediate fluorescence yield plateau level - F_max maximum fluorescence yield - F_i mitial fluorescence yield increase from F_o to F_pl(F_pl-F_o) - F_v total variable fluorescence yield (F_max-F_o) - DCMU dichlorophenyl-dimethylurea     

Photosystem II heterogeneity: the acceptor side

It is well known that two photosystems, I and II, are needed to transfer electrons from H₂O to NADP⁺ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS II and PS II units, (b) RC complex containing electron acceptor Q₁ or Q₂; and (c) electron acceptor complex: Q_A (having two different redox potentials Q_L and Q_H) and Q_B (Q_B-type; Q'_B type; and non-Q_B type); additional components such as iron (Q-400), U (E_m,7=–450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.Abbreviations DCBQ 2,5 or 2,6 dichloro-p-benzoquinone - DMQ dimethylquinone - Q_A primary plastoquinone electron acceptor of photosystem II - Q_B secondary plastoquinone electron acceptor of photosystem II - IODP successive fluorescence levels during time course of chlorophyll a fluorescence: O for origin, I for inflection, D for dip or plateau, and P for peak     

Development of photosystem-II activity during irradiance of etiolated Helianthus (Asteraceae) seedlings

Light-induced development of photosystem (PS)-II activity was followed during irradiance of etiolated Helianthus annuus (sunflower) cotyledons using chlorophyll a fluorescence. Cotyledons from seedlings grown in continuous darkness for 6 d were exposed to 100 μmol photons·m⁻²·s⁻¹ for time periods of 1, 3, 6, and 12 h. Associated with increased time of irradiance exposure were significant: (1) increases in concentration of PS II, (2) increases in quantum efficiency of PS II, (3) decreases in the ratio of PS-II quinone_B (Q_B)-nonreducing centers to total PS-II centers (PS-II Q_B-nonreducing centers + PS-II Q_B-reducing centers), and (4) decreases in the ratio of slow PS-II Q_B-reducing centers to total PS-II Q_B-reducing centers (fast PS-II Q_B-reducing centers + slow PS-II Q_B-reducing centers). The results support the hypotheses that development of PS II involves assembly of complexes which initially cannot reduce Q_B and that heterogeneous aspects of PS-II pools during chloroplast maturation may represent different developmental states.     

Probing of photosynthetic reactions in four phytoplanktonic algae with a PEA fluorometer

High-resolution light-induced kinetics of chlorophyll fluorescence (OJIP transients) were recorded and analyzed in cultures of diatoms (Thalassiosira weissflogii, Chaetoceros mulleri) and dinoflagellates (Amphidinium carterae, Prorocentrum minimum). Fluorescence transients showed the rapid exponential initial rise from the point O indicating low connectivity between PS II units and high absorption cross-section of PS II antenna. Dark-adapted dinoflagellates revealed capability to maintain the PS I-mediated re-oxidation of the PQ pool at the exposure to strong actinic light that may lead to the underestimation of F _M value. In OJIP transients recorded in phytoplanktonic algae the fluorescence yield at the point O exceeded F _O level because Q_A has been already partly reduced at 50 μs after the illumination onset. PEA was also employed to study the recovery of photosynthetic reactions in T. weissflogii during incubation of nitrogen starved cells in N-replete medium. N limitation caused the impairment of electron transport between Q_A and PQs, accumulation of closed PS II centers, and the reduced ability to generate transmembrane ΔpH upon illumination, almost fully restored during the recovery period. The recovered cells showed much higher values of NPQ than control ones suggesting maximization of photoprotection mechanisms in the population with a ‘stress history.’     

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that Q_A of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of Q_A than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.Abbreviations pBQ p-benzoquinone - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCIP 2,6-dichloroindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DQ duroquinone(tetramethyl-p-benzoquinone) - FeCN ferricyanide (potassium hexacyanoferrat) - MV methylviologen - NADPH,NADP⁺ reduced or oxidized form of nicotinamide adenine dinucleotide phosphate respectively - PpBQ phenyl-p-benzoquinone - PQ plastoquinone - PS II photosystem II - PS I photosystem I - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - E microEinstein     

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

The gdh and gdhr genes, encoding B₁₂-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly₄Ser)₄ linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in PS II heterogeneity in response to osmotic and ionic stress in wheat leaves (Triticum aestivum)

High salt stress involves ionic stress as well as osmotic stress. In this work we have tried to differentiate between the ionic and osmotic effects of salt stress on the basis of their ability to cause changes in PS II heterogeneity. PS II heterogeneity is found to vary with environmental conditions. Osmotic stress caused no change in the Q(B) reducing side heterogeneity and a reversible change in antenna heterogeneity. The number of Q(B) non-reducing centers increased under ionic stress but were unaffected by osmotic stress. On the other hand ionic stress led to a partially irreversible change in Q(B) reducing side heterogeneity and a reversible change in antenna heterogeneity. In response to both ionic and osmotic effect, there is conversion of active PS IIα centres to inactive PSIIβ and γ centres.     

Evolution of PS IIα and PS IIβ centers during the greening of Euglena gracilis Z: Correlations with changes in lipid content

The building up of the two types of reaction centers, PS II and PS II, was investigated during the greening of Euglena gracilis Z cells in resting medium. The maximal values in the proportion of PS II centers (55%) and in the oxygen evolved per chlorophyll were reached at the outbreak of greening, when accumulation of galactolipids (MGDG and DGDG) rich in unsaturated fatty acids occurred, and when anionic lipids (SQDG and PG) emerged. As the greening progressed, the chlorophyll accumulation corresponded to a secondary enrichment in PS II centers, which built up more rapidly than PS II centers; correlatively, a general saturation of the fatty acids constitutive of all lipid classes took place.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DGDG digalactosyldiacylglycerol - FAME Tatty acid methyl esters - HEPES acide (N-[2-hydroxyethyl]piperazine-N-[2-ethane sulfonic] - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PQ plastoquinone - PS I Photosystem I - PS II Photosystem II - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - SQDG sulfoquinovosyldiacylglycerol     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with Photosystem II

Chlorophyll a fluorescence induction (FI) is widely used as a probe for studying photosynthesis. On illumination, fluorescence emission rises from an initial level O to a maximum P through transient steps, termed J and I. FI kinetics reflect the overall performance of photosystem II (PSII). Although FI kinetics are commonly and easily measured, there is a lack of consensus as to what controls the characteristic series of transients, partially because most of the current models of FI focus on subsets of reactions of PSII, but not the whole. Here we present a model of fluorescence induction, which includes all discrete energy and electron transfer steps in and around PSII, avoiding any assumptions about what is critical to obtaining O J I P kinetics. This model successfully simulates the observed kinetics of fluorescence induction including O J I P transients. The fluorescence emission in this model was calculated directly from the amount of excited singlet-state chlorophyll in the core and peripheral antennae of PSII. Electron and energy transfer were simulated by a series of linked differential equations. A variable step numerical integration procedure (ode15s) from MATLAB provided a computationally efficient method of solving these linked equations. This in silico representation of the complete molecular system provides an experimental workbench for testing hypotheses as to the underlying mechanism controlling the O J I P kinetics and fluorescence emission at these points. Simulations based on this model showed that J corresponds to the peak concentrations of Q _A ⁻ Q_B (Q_A and Q_B are the first and second quinone electron acceptor of PSII respectively) and Q _A ⁻ Q _B ⁻ and I to the first shoulder in the increase in concentration of Q _A ⁻ Q _B ²⁻ . The P peak coincides with maximum concentrations of both Q _A ⁻ Q _B ²⁻ and PQH₂. In addition, simulations using this model suggest that different ratios of the peripheral antenna and core antenna lead to differences in fluorescence emission at O without affecting fluorescence emission at J, I and P. An increase in the concentration of Q_B-nonreducing PSII centers leads to higher fluorescence emission at O and correspondingly decreases the variable to maximum fluorescence ratio (F _v/F _m).     

Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers

Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, Q_A, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320–330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution in the presence of DCBQ or DMBQ support the conclusion that inactive Photosystem II complexes have a functional water oxidation system.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - F_o initial fluorescence level using dark-adapted thylakoids - Inactive reaction centers reaction centers inactive in plastoquinone reduction - PS II Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II Department of Plant Biology, University of IllinoisDepartment of Physiology & Biophysics, University of Illinois     

Photophysical processes of energy conversion in thylakoid membranes of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> mutants D1-R323H,D1-R323D,and D1-R323L

Chlamydomonas reinhardtii mutants D1-R323H, D1-R323D, and D1-R323L showed elevated chlorophyll fluorescence yields, which increased with decline of oxygen evolving capacity. The extra step K ascribed to the disturbance of electron transport at the donor side of PS II was observed in OJIP kinetics measured in mutants with a PEA fluorometer. Fluorescence decay kinetics were recorded and analyzed in a pseudo-wild type (pWt) and in mutants of C. reinhardtii with a Becker and Hickl single photon counting system in pico- to nanosecond time range. The kinetics curves were fitted by three exponentials. The first one (rapid, with lifetime about 300 ps) reflects energy migration from antenna complex to the reaction center (RC) of photosystem II (PS II); the second component (600–700 ps) has been assigned to an electron transfer from P680 to Q_A, while the third one (slow, 3 ns) assumingly originates from charge recombination in the radical pair [P680^+• Pheo^−•] and/or from antenna complexes energetically disconnected from RC II. Mutants showed reduced contribution of the first component, whereas the yield of the second component increased due to slowing down of the electron transport to Q_A. The mutant D1-R323L with completely inactive oxygen evolving complex did not reveal rapid component at all, while its kinetics was approximated by two slow components with lifetimes of about 2 and 3 ns. These may be due to two reasons: a) disconnection between antennae complexes and RC II, and b) recombination in a radical pair [P680^+• Pheo^−•] under restricted electron transport to Q_A. The data obtained suggest that disturbance of oxygen evolving function in mutants may induce an upshift of the midpoint redox potential of Q_A/Q_A⁻ couple causing limitation of electron transport at the acceptor side of PS II.     

Pair-wise interactions of polymerization inhibitory contact site mutations of hemoglobin-S

The linkage of pair-wise interactions of contact site mutations of HbS has been studied using Le Lamentin [His-20 (α)→Gln], Hoshida [Glu-43 (β)→Gln] and α₂β₂^T87Q mutations as the prototype of three distinct classes of contact sites of deoxy HbS fiber. Binary mixture experiments established that β^A-chain with the Thr-87 (β)→Gln mutation is as potent as the γ-chain of HbF (α₂γ₂) in inhibiting polymerization. On combining the influence of Le Lamentin mutation with that of β₂^T87Q mutations; the net influence is only partial additivity. On the other hand, in binary mixture studies, combined influence of Hoshida mutation with that of β₂^T87Q mutations is synergistic. Besides, a significant level of synergistic complementation is also seen when the Le Lamentin and Hoshida mutations are combined in HbS (symmetrical tetramers). Le Lamentin and Hoshida mutation introduced into the cis-dimer of the asymmetric hybrid tetramer completely neutralizes the Val-6 (β) dependent polymerization. Accordingly, we propose that combining the perturbation of intra-double strand contact site with that of an inter-double strand contact site exhibit synergy when they are present in two different chains of the αβ dimer. A comparison of the present results with that of the earlier studies suggest that when the two contact site perturbations are from the same sub-unit of the αβ dimer only partial additivity is observed. The map of interaction linkage of the contact site mutations exposes new strategies in the design of novel anti-sickling Hbs for the gene therapy of sickle cell disease.     

Characterization of the photosystem II centers inactive in plastoquinone reduction by fluorescence induction

In order to characterize the photosystem II (PS II) centers which are inactive in plastoquinone reduction, the initial variable fluorescence rise from the non-variable fluorescence level F_o to an intermediate plateau level F_i has been studied. We find that the initial fluorescence rise is a monophasic exponential function of time. Its rate constant is similar to the initial rate of the fastest phase (-phase) of the fluorescence induction curve from DCMU-poisoned chloroplasts. In addition, the initial fluorescence rise and the -phase have the following common properties: their rate constants vary linearly with excitation light intensity and their fluorescence yields are lowered by removal of Mg⁺⁺ from the suspension medium. We suggest that the inactive PS II centers, which give rise to the fluorescence rise from F_o to F_i, belong to the -type PS II centers. However, since these inactive centers do not display sigmoidicity in fluorescence, they thus do not allow energy transfer between PS II units like PS II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DMQ 2,5-dimethyl-p-benzoquinone - F_o initial non-variable fluorescence yield - F_m maximum fluorescence yield - F_i intermediate fluorescence yield - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

GABA Analogues Derived from 4-Aminocyclopent-1-enecarboxylic Acid

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA_A agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the α,β-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant α₁β₂ γ_2L GABA_A receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC₅₀ of 42 μM for the inhibition of a GABA EC₅₀ dose (50 μM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the α₁β₂γ_2L GABA_A receptor. Special issue article in honour of Dr. Graham Johnston.     

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

GABA_A receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABA_A receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (E_Cl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics.     

Stiffness of γ subunit of F1-ATPase

F₁-ATPase is a molecular motor in which the γ subunit rotates inside the α₃β₃ ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F₁-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F₁ and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F₁ from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α₃β₃ ring, and the complex of the external part of the γ subunit and the α₃β₃ ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F_o and F₁-ATPase.     

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B     

Structural and functional characterization of <Emphasis Type="Italic">Delphinus delphis</Emphasis> hemoglobin system

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC–electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major β (β1 16,022 Da, β2 16,036 Da, β3 16,036 Da, labeled according to their progressive elution times) and two major α globins (α1 15,345 Da, α2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that β2 globin differs from β1 for the substitution Val126 → Leu, while β3 globin differs from β2 for the isobaric substitution Lys65 → Gln. The α2 globin differs from the α1 for the substitution Ser15 → Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC–ESI-MS analysis revealed that the fraction with higher pI (HbI) contained β1, β2 and both the α globins, and the fraction with lower pI (HbII) contained β3 and both the α globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA₀, with HbII showing the more pronounced differences. With respect to HbA₀, either the substitution Proβ5 → Gly or the Proβ5 → Ala is present in all the cetacean β globins sequenced so far, and it has been hypothesized that position 5 of β globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lysβ65 → Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA₀.