Positions of QAand ChlZRelative to Tyrosine YZand YDin Photosystem II Studied by Pulsed EPR

PELDOR (Pulsed Electron eLectron DOuble Resonance) was applied to determinethe distance of between Y_Zand Q_A ^-inY_D-less mutant of Chlamydomonas reinhardtiiin Tris-treatedand Zn-substituted preparation of photosystem II. The value of distance wasfound to be 34.5 ± 1 Â. A 2+1 electron spin echo method has beenapplied to measure the orientation of the radius-vector RfomY_Dto Chl_Zin a membrane-oriented photosystem II. The anglebetween Rand the membrane normal nwas determined to be 50 ±5^°, using the distance 29.4 ± 0.5 Â determined in non-orientedPS II.     

Minor but key chlorophylls in Photosystem II

A ‘metal-free’ chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

Pigment protein complexes and functional properties of tetratype resulting from crosses between CP1 and CP2 less Chlamydomonas mutants

A chlorophyll b-less mutant of Chlamydomonas reinhardtii (Pg ₂₇) was isolated after UV irradiation of the wild type cells. This photosynthetically competent mutant totally lacks chlorophyll b and the CP₂ chlorophyll-protein complex. However, SDS-PAGE, proteolytic digestions and immunodetections demonstrated that the 24–25 Kd apoproteins of the lacking CP₂ complex are still present in thylakoids of the Pg₂₇ mutant. It is concluded that this CP₂-less mutant is affected in the biosynthesis pathway of chlorophyll b.This CP₂-less mutant was crossed with a CP₁-less mutant (Fl₅) Fluorescence emission spectra and fluorescence inductions in the presence of DCMU were analysed in the resulting (cp ₂ ^– , cp ₁ ⁺ ), (cp ₂ ⁺ , cp ₁ ^– ), (cp ₂ ⁺ , cp ₁ ⁺ ), cp ₂ ^– , cp ₁ ^– )tetratype. Differences in PS 2 optical cross section and in the relative amplitude or localisation of fluorescence emission peaks fit well with a quadripartite model where PS1 and PS2 would each correspond to a reaction centre core complex (CP₁ and CP₂ respectively) associated to a light harvesting antenna (LHC1 and LHC2 respectively). The occurrence of energy transfers from PS1 peripheral antenna to PS2 in the Fl ₅ mutant shows that, in absence of CP₁, at least a part of its associated PS1 light harvesting antenna migrates in the PS2 containing appressed thylakoids.Abbreviations Chl Chlorophyll - LHC Light harvesting chl a/b complex - CP₂ Predominant form of LHC or SDS polyacrylamide gels - WT Wild type - DM Double mutant (cp ₁ ^– , cp ₂ ^– ) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - DOC-PAGE Deoxycholate polyacrylamide gel electrophoresis     

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P₆₈₀⁺Q_A⁻ limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P₆₈₀⁺ by the secondary electron donor Tyrosine Z cannot occur because Y_Z is already oxidized. The 1.4 ms recombination is seen in Y_Z-less mutants of the cyanobacterium Synechocystis. However, the rate of P₆₈₀⁺Q_A⁻ recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P₆₈₀⁺ reduction by Y_Z were obtained, and the rate of the competing P₆₈₀⁺Q_A⁻ recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y_Z^ox slightly decreased the efficiency of excitation trapping but did not seem to accelerate P₆₈₀⁺Q_A⁻ recombination. The two P₆₈₀⁺Q_A⁻ lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

Correlation of the cytochrome c 2 content of cyanobacterial Photosystem II with the EPR properties of the oxygen-evolving complex

The Mn₄ cluster of PS II advances through a series of oxidation states (S states) that catalyze the breakdown of water to dioxygen in the oxygen-evolving complex. The present study describes the engineering and purification of highly active PS II complexes from mesophilic His-tagged Synechocystis PCC 6803 and purification of PS II core complexes from thermophilic wild-type Synechococcus lividus with high levels of the extrinsic polypeptide, cytochrome c ₅₅₀. The g = 4.1 S₂ state EPR signal, previously not characterized in untreated cyanobacterial PS II, is detected in high yields in these PS II preparations. We present a complete characterization of the g = 4.1 state in cyanobacterial His-tagged Synechocystis PCC 6803 PS II and S. lividus PS II. Also presented are a determination of the stoichiometry of cytochrome c ₅₅₀ bound to His-tagged Synechocystis PCC 6803 PS II and analytical ultracentrifugation results which indicate that cytochrome c ₅₅₀ is a monomer in solution. The temperature-dependent multiline to g = 4.1 EPR signal conversion observed for the S₂ state in cyanobacterial PS II with high cytochrome c ₅₅₀ content is very similar to that previously found for spinach PS II. In spinach PS II, the formation of the S₂ state g = 4.1 EPR signal has been found to correlate with the binding of the extrinsic 17 and 23 kDa polypeptides. The finding of a similar correlation in cyanobacterial PS II with the binding of cytochrome c ₅₅₀ suggests a functional homology between cytochrome c ₅₅₀ and the 17 and 23 kDa extrinsic proteins of spinach PS II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Multiple sites of retardation of electron transfer in Photosystem II after hydrolysis of phosphatidylglycerol

Phosphatidylglycerol (PG), containing the unique fatty acid Δ3, trans-16:1-hexadecenoic acid, is a minor but ubiquitous lipid component of thylakoid membranes of chloroplasts and cyanobacteria. We investigated its role in electron transfers and structural organization of Photosystem II (PSII) by treating Arabidopsis thaliana thylakoids with phospholipase A₂ to decrease the PG content. Phospholipase A₂ treatment of thylakoids (a) inhibited electron transfer from the primary quinone acceptor Q_A to the secondary quinone acceptor Q_B, (b) retarded electron transfer from the manganese cluster to the redox-active tyrosine Z, (c) decreased the extent of flash-induced oxidation of tyrosine Z and dark-stable tyrosine D in parallel, and (d) inhibited PSII reaction centres such that electron flow to silicomolybdate in continuous light was inhibited. In addition, phospholipase A₂ treatment of thylakoids caused the partial dissociation of (a) PSII supercomplexes into PSII dimers that do not have the complete light-harvesting complex of PSII (LHCII); (b) PSII dimers into monomers; and (c) trimers of LHCII into monomers. Thus, removal of PG by phospholipase A₂ brings about profound structural changes in PSII, leading to inhibition/retardation of electron transfer on the donor side, in the reaction centre, and on the acceptor side. Our results broaden the simple view of the predominant effect being on the Q_B-binding site.     

Direct quantification of the four individual S states in Photosystem II using EPR spectroscopy

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn₄ cluster are known in all S states except S₄. Many signals are insufficiently understood and the S₀, S₁, and S₃ states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S₀, S₁, and S₃ states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S₀, split S₁, split S₃ and S₂ state multiline EPR signals. We demonstrate that quantification of the S₁, S₃ and S₀ states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S₁ → S₂ transition proceeds without misses following a saturating flash at 1 °C, whilst substantial misses occur in the S₂ → S₃ transition following the second flash.     

Temperature-dependent changes in Photosystem II heterogeneity support a cycle of Photosystem II during photoinhibition

High light treatments were given to attached leaves of pumpkin (Cucurbita pepo L.) at room temperature and at 1°C where the diffusion- and enzyme-dependent repair processes of Photosystem II are at a minimum. After treatments, electron transfer activities and fluorescence induction were measured from thylakoids isolated from the treated leaves. When the photoinhibition treatment was given at 1°C, the Photosystem II electron transfer assays that were designed to require electron transfer to the plastoquinone pool showed greater inhibition than electron transfer from H₂O to paraphenyl-benzoquinone, which measures all PS II centers. When the light treatment was given at room temperature, electron transfer from H₂O to paraphenyl-benzoquinone was inhibited more than whole-chain electron transfer. Variable fluorescence measured in the presence of ferricyanide decreased only during room-temperature treatments. These results suggest that reaction centers of one pool of Photosystem II, non-Q_B-PS II, replace photoinhibited reaction centers at room temperature, while no replacement occurs at 1°C. A simulation of photoinhibition at 1°C supports this conclusion.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCMU 3-(3,4,-dichlorophenyl)-1,1,-dimethylurea - DCPIP dichlorophenol-indophenol (2,6-dichloro-4((4-hydroxyphenyl)imino)-2,5-cyclohexadien-1-one) - DPC diphenyl carbazide (2,2-diphenylcarbonic dihydrazide) - FeCN ferricyanide (hexacyanoferrate(III)) - _app apparent quantum yield of photosynthetic oxygen evolution - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - PPBQ phenyl-p-benzoquinone - PPFD photosynthetic photon flux density - PQ pool plastoquinone - Q_B secondary quinone acceptor of PS II - RT room temperature - WC whole chain electron transfer     

Key cofactors of Photosystem II cores from four organisms identified by 1.7-K absorption, CD and MCD

Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98–103]. Electrochromic shifts resulting from Q_A reduction by 1.7-K illumination were recorded, and the Q_x and Q_y absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Q_x transition is ∼3 nm (100 cm⁻¹) to higher energy in cyanobacteria than in the plants. The predominant Q_y shift appears in the range 683–686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Q_y region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.     

The origin of split EPR signals in the Ca2+-depleted Photosystem II

A light-driven reaction model for the Ca²⁺-depleted Photosystem (PS) II is proposed to explain the split signal observed in electron paramagnetic resonance (EPR) spectra based on a comparison of EPR assignments with recent x-ray structural data. The split signal has a splitting linewidth of 160 G at around g = 2 and is seen upon illumination of the Ca²⁺-depleted PS II in the S₂ state associated with complete or partial disappearance of the S₂ state multiline signal. Another g=2 broad ESR signal with a 110 G linewidth was produced by 245 K illumination for a short period in the Ca²⁺-depleted PS II in S₁ state. At the same time a normal Y_Z^· radical signal was also efficiently trapped. The g=2 broad signal is attributed to an intermediate S₁X^· state in equilibrium with the trapped Y_Z^· radical. Comparison with x-ray structural data suggests that one of the split signals (doublet signal) is attributable to interaction between His 190 and the Y_Z^· radical, and other signals is attributable to interaction between His 337 and the manganese cluster, providing further clues as to the mechanism of water oxidation in photosynthetic oxygen evolution.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca²⁺-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y_Z. The ratio of the intensities between the g=2 broad signal and the Y_Z signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y_Z signal decayed together in parallel with the appearance of the S₂ state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S₁X state in the transition from the S₁ to the S₂ state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y_Z. Possible reactions in the S state transitions in Ca²⁺-depleted PS II were discussed.     

Reconstitution of photosynthetic charge accumulation and oxygen evolution in CaCl2-treated PS II particles: II: EPR evidence for reactivation of the S1 → S2 transition in CaCl2-treated PS II particles with the 17-, 23-, and 34-kDa proteins

Extraction of PS II particles with 1 M CaCl₂ caused complete disappearance of the light-induced signal of the possible Kok S₂ state of the water-splitting complex and total loss of the O₂, evolving activity, concomitant with perfect removal of the 17-, 23- and 34-kDa proteins from the particles. The recovery of the multiline signal in the CaCl₂-treated PS II was performed by reinserting the 34-kDa protein, when CI^? was present in the solution for the EPR measurement. However, in the absence of Cl^?, besides the 34-kDa protein, the 17- and 23-kDa proteins were required for the recovery of the signal. These results are compared with the results on the recovery of the O₂, evolution in the reconstituted PS II to examine the role of these three proteins on the water splitting.     

Performance of active Photosystem II centers in photoinhibited pea leaves

Effects of photoinhibition on photosynthesis in pea (Pisum sativum L.) leaves were investigated by studying the relationship between the severity of a photoinhibitory treatment (measured as F_v/F_m) and several photoacoustic and chlorophyll a fluorescence parameters. Because of the observed linear relationship between the decline of F_v/F_m and the potential oxygen evolution rate determined by the photoacoustic method, the parameter F_v/F_m was used as an indicator for the severity of photoinhibition. Our analysis revealed that part of the Photosystem II (PS II) reaction centers is inactive in oxygen evolution and is also less sensitive to photoinhibition. Correcting the parameter q_P (fraction of open PS II reaction centers) for inactive PS II centers unveiled a strong increase of q_P in severely inhibited pea leaves, indicating that the inactivated active centers do no longer contribute to q_P and that photoinhibition has an all or none effect on PS II centers. Analysis of q_E (energy quenching) demonstrated its initial increase possibly associated with dephosphorylation of LHC II. Analysis of q_I (photoinhibition dependent quenching) showed that the half-time of recovery of q_I increases steeply below an F_v/F_m of 0.65. This increase of the relaxation half-time corresponds with a decrease of the electron transport rate J and tentatively indicates that the supply of ATP, needed for the recovery, starts to decrease. The data indicate the necessity of correcting for inactive centers in order to make valuable conclusions about effects of photoinhibition on photosynthetic parameters.     

Study on the photo-generation of superoxide radicals in Photosystem II with EPR spin trapping techniques

Direct EPR evidence of the photo-generation of superoxide radicals (O₂ ^–.) was obtained by using a novel spin trapping probe in spinach Photosystem II (PS II) membrane fragments. The production of O₂ ^–. was detected by following the formation of 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) superoxide adducts (DEPMPO-OOH). The inhibition of O₂ ^–. formation by 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU) and the 77 K fluorescence spectrum indicated that O₂ ^–. were generated from PS II, not from PS I. The inhibition of O₂ ^–. formation by DCMU also suggested that O₂ ^–. were generated from the Q_Bbinding site, not at a site prior to DCMU blockage. The extrinsic proteins and Mn are very important to eliminate O₂ ^–., showing that the oxygen-evolving system is involved in O₂ ^–. removal rather than production.This revised version was published online in October 2005 with corrections to the Cover Date.     

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina¹⁰⁴) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light. It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of Q_A reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flash During greening in continuous light of IL pea leaves, an inhibitory effect on Q_A photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.     

Very high light resistant mutants of Chlamydomonas reinhardtii: Responses of Photosystem II,nonphotochemical quenching and xanthophyll pigments to light and CO2

We have isolated very high light resistant nuclear mutants (VHL ^R) in Chlamydomonas reinhardtii, that grow in 1500–2000 mol photons m^–2 s^–1 (VHL) lethal to wildtype. Four nonallelic mutants have been characterized in terms of Photosystem II (PS II) function, nonphotochemical quenching (NPQ) and xanthophyll pigments in relation to acclimation and survival under light stress. In one class of VHL ^R mutants isolated from wild type (S4 and S9), VHL resistance was accompanied by slower PS II electron transfer, reduced connectivity between PS II centers and decreased PS II efficiency. These lesions in PS II function were already present in the herbicide resistant D1 mutant A251L (L ^*) from which another class of VHL ^R mutants (L4 and L30) were isolated, confirming that optimal PS II function was not critical for survival in very high light. Survival of all four VHL ^R mutants was independent of CO₂ availability, whereas photoprotective processes were not. The de-epoxidation state (DPS) of the xanthophyll cycle pigments in high light (HL, 600 mol photons m^–2 s^–1) was strongly depressed when all genotypes were grown in 5% CO₂. In S4 and S9 grown in air under HL and VHL, high DPS was well correlated with high NPQ. However when the same genotypes were grown in 5% CO₂, high DPS did not result in high NPQ, probably because high photosynthetic rates decreased thylakoid pH. Although high NPQ lowered the reduction state of PS II in air compared to 5% CO₂ at HL in wildtype, S4 and S9, this did not occur during growth of S4 and S9 in VHL. L ^* and VHL ^R mutants L4 and L30, also showed high DPS with low NPQ when grown air or 5% CO₂, possibly because they were unable to maintain sufficiently high pH due to constitutively impaired PS II electron transport. Although dissipation of excess photon energy through NPQ may contribute to VHL resistance, there is little evidence that the different genes conferring the VHL ^R phenotype affect this form of photoprotection. Rather, the decline of chlorophyll per biomass in all VHL ^R mutants grown under VHL suggests these genes may be involved in regulating antenna components and photosystem stoichiometries.This revised version was published online in October 2005 with corrections to the Cover Date.     

The S1 split signal of photosystem II; a tyrosine-manganese coupled interaction

Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q_A⁻ is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (< 5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5-20 K. An optically silent electron donor to P680⁺ is observed with the same decay kinetics as the S₁ split signal. Cryogenic donors to P680⁺ seen are: (i) transient (t_1/2 ∼ 150 s) tyrosine related species, including ‘split signals’ (∼ 15% total centers), (ii) reduced cytochrome b₅₅₉ (∼ 30-50% centers), and (iii) an organic donor, possibly an amino acid side chain, (∼ 30% centers).     

Consequences of LHC II deficiency for photosynthetic regulation in chlorina mutants of barley

The light-harvesting chlorophyll a/b proteins associated with PS II (LHC II) are often considered to have a regulatory role in photosynthesis. The photosynthetic responses of four chlorina mutants of barley, which are deficient in LHC II to varying degrees, are examined to evaluate whether LHC II plays a regulatory role in photosynthesis. The efficiencies of light use for PS I and PS II photochemistry and for CO₂ assimilation in leaves of the mutants were monitored simultaneously over a wide range of photon flux densities of white light in the presence and absence of supplementary red light. It is demonstrated that the depletions of LHC II in these mutants results in a severe imbalance in the relative rates of excitation of PS I and PS II in favour of PS I, which cannot be alleviated by preferential excitation of PS II. Analyses of xanthophyll cycle pigments and fluorescence quenching in leaves of the mutants indicated that the major LHC II components are not required to facilitate the light-induced quenching associated with zeaxanthin formation. It is concluded that LHC II is important to balance the distribution of excitation energy between PS I and PS II populations over a wide range of photon flux densities. It appears that LHC II may also be important in determining the quantum efficiency of PS II photochemistry by reducing the rate of quenching of excitation energy in the PS II primary antennae.Abbreviations Fm, Fv maximal and variable fluorescence yields in a light adapted state - LHC II light harvesting chlorophyll a/b protein complex associated with PS II - q_p photochemical quenching - A₈₂₀ light-induced absorbance change at 820 nm - ø_PSI, ø_PSII relative quantum efficiencies of PS I and PS II photochemistry - øCO₂ quantum yield of CO₂ assimilation     

The antagonism between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated biological responses induced by 1alpha,25-dihydroxyvitamin D3 assessed by NB4 cell differentiation

We synthesized all eight possible A-ring diastereomers of 2-methyl substituted analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and also all eight A-ring diastereomers of 2-methyl-20-epi-1alpha,25(OH)2D3. Their biological activities, especially the antagonistic effect on non-genomic pathway-mediated responses induced by 1alpha,25(OH)2D3 or its 6-s-cis-conformer analog, 1alpha,25(OH)2-lumisterol3, were assessed using an NB4 cell differentiation system. Antagonistic activity was observed for the 1beta-hydroxyl diastereomers, including 2beta-methyl-1beta,25(OH)2D3 and 2beta-methyl-3-epi-1beta,25(OH)2D3. Very interestingly, 2beta-methyl-3-epi-1alpha,25(OH)2D3 also antagonized the non-genomic pathway, despite its 1alpha-hydroxyl group. Other 1alpha-hydroxyl diastereomers did not show antagonistic activity. 20-epimerization diminished the antagonistic effect of all of these analogs on the non-genomic pathway. These findings suggested that the combination of the 2-methyl substitution of the A-ring and 20-epimerization of the side chain could alter the biological activities in terms of antagonism of non-genomic pathway-mediated biological response. Based on a previous report, 2-methyl substitution alters the equilibrium of the A-ring conformation between the alpha- and beta-chair conformers. The 2beta-methyl diastereomers, which exhibited antagonism on non-genomic pathway-mediated response, were considered to prefer the beta-conformer. Further examination to elucidate the relationship between the altered ligand shape and receptors interaction will be important for molecular level understanding of the mechanism of antagonism of the non-genomic pathway.