Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyl transferase: the role of N341, Y60 and F351 in tetrahydrofolate binding.

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10 -methylene THE SHMT also catalyses several THF-independent side reactions such as cleavage of P-hydroxy amino acids, trans-amination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect oil either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a Mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three Mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Photocytotoxic ferrocene-appended (L-tyrosine)copper(II) complexes of phenanthroline bases

Copper(II) complexes of ferrocene(Fc)-conjugated reduced Schiff base of L-tyrosine (Fc-TyrH), viz., Cu(Fc-Tyr)(L)](ClO4), where L is 1,10-phenanthroline (phen, 1), dipyrido3,2-d:2',3'-f]quinoxaline (dpq, 2), dipyrido3,2-a:2',3'-c]phenazine (dppz, 3) and 2-(naphthalen-1-yl)-1H-imidazo4,5-f]1,10]phenanthroline (nip, 4), were prepared and tested for their photocytotoxicity in cancer cells. Cu(Fc-Phe)(phen)](-ClO4) (5) of L-phenylalanine and Cu(Ph-Tyr)(L)(ClO4)] of the reduced Schiff base Ph-TyrH derived from benzaldehyde and L-tyrosine having phen (6) and dppz (7), and Cu(Ph-Phe)(phen)(ClO4)] (8) using L-phenylalanine were prepared and used as controls. Complexes 5 and 6 were structurally characterized by X-ray crystallography. A copper(II)-based d-d band near 600 nm and a ferrocenyl band at similar to 450 nm were observed in DMF-Tris-HCI buffer (1:4 v/v) in respective complexes. The complexes are photocleavers of pUC19 DNA in visible light forming (OH)-O-center dot radicals. They are cytotoxic in HeLa (human cervical cancer) and MCF-7 (human breast cancer) cells showing an enhancement of cytotoxicity in visible light. Fluorescence imaging shows nuclear localization of the complexes.

Analysis of designed beta-hairpin peptides: molecular conformation and packing in crystals

The crystal structures of several designed peptide hairpins have been determined in order to establish features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has been induced using a centrally positioned (D)Pro-Xxx segment (Xxx = (L)Pro, Aib, Ac(6)c, Ala; Aib = alpha-aminoisobutyric acid; Ac(6)c = 1-aminocyclohexane-1-carboxylic acid). Structures of the peptides Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (2, polymorphic forms labeled as 2a and 2b), Boc-Leu-Val-Val-(D)Pro-(L)Pro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-Val-(D)Pro-Aib-Leu-Phe-Val-OMe (4, polymorphic forms labeled as 4a and 4b), Boc-Leu-Phe-Val-(D)Pro-Ac(6)c-Leu-Phe-Val-OMe (5) and Boc-Leu-Phe-Val-(D)Pro-Ala-Leu-Phe-Val-OMe (6) are described. All the octapeptides adopt type II' beta-turn nucleated hairpins, stabilized by three or four cross-strand intramolecular hydrogen bonds. The angle of twist between the two antiparallel strands lies in the range of -9.8 degrees to -26.7 degrees. A detailed analysis of packing motifs in peptide hairpin crystals is presented, revealing three broad modes of association: parallel packing, antiparallel packing and orthogonal packing. An attempt to correlate aggregation modes in solution with observed packing motifs in crystals has been made by indexing of crystal faces in the case of three of the peptide hairpins. The observed modes of hairpin aggregation may be of relevance in modeling multiple modes of association, which may provide insights into the structure of insoluble polypeptide aggregates.

Stabilizing effect of electrostatic vs. aromatic interactions in diproline nucleated peptide beta-hairpins

The contribution of Tyr-His vs. Cys-His interacting pairs to the scaffold stability of (D)Pro-(L)Pro nucleated peptide beta-hairpins has been examined. We present direct evidence for the superiority of the Cys-His pairs, mediated by sulphur-imidazole interactions, as added stabilizing agents of the beta-hairpin scaffold.

Conformational Diversity in Contryphans from Conus Venom: cis-trans Isomerisation and Aromatic/Proline Interactions in the 23-Membered Ring of a 7-Residue Peptide Disulfide Loop

Conformational diversity or shapeshifting in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

Ferrocenyl-L-amino acid copper(II) complexes showing remarkable photo-induced anticancer activity in visible light

Ferrocene-conjugated copper(II) complexes Cu(Fc-aa)(aip)](ClO4) (1-3) and (Cu(Fc-aa)(pyip)](ClO4) (4-6) of L-amino acid reduced Schiff bases (Fc-aa), 2-(9-anthryl)-1H-imidazo4,5-f]1,10]phenanthroline (aip) and 2-(1-pyrenyl)-1H-imidazo4,5-f] 1,10]phenanthroline (pyip), where Fc-aa is ferrocenylmethyl-L-tyrosine (Fc-Tyr in 1, 4), ferrocenylmethyl-L-tryptophan (Fc-Trp in 2, 5) and ferrocenylmethyl-L-methionine (Fc-Met in 3, 6), were prepared and characterized, and their photocytotoxicity was studied (Fc = ferrocenyl moiety). Phenyl analogues, viz. (Cu(Ph-Met)(aip)](ClO4) (7) and (Cu(Ph-Met)(pyip)](ClO4) (8), were prepared and used as control compounds. The bis-imidazophenanthroline copper(II) complexes, viz. (Cu(aip)(2)(NO3)](NO3) (9) and Cu(pyip)(2)(NO3)](NO3) (10), were also prepared and used as controls. Complexes 1-6 having a redox inactive cooper(II) center showed the Fc(+)-Fc redox couple at similar to 0.5 V vs. SCE in DMF-0.1 mol (Bu4N)-N-n](ClO4). The copper(II)-based d-d band was observed near 600 nm in DMF-Tris-HCl buffer (1 :1 v/v). The ferrocenyl complexes showed low dark toxicity, but remarkably high photocytotoxicity in human cervical HeLa and human breast adenocarcinoma MCF-7 cancer cells giving an excellent photo-dynamic effect while their phenyl analogues were inactive. The photo-exposure caused significant morphological changes in the cancer cells when compared to the non-irradiated ones. The photophysical processes were rationalized from the theoretical studies. Fluorescence microscopic images showed 3 and 6 localizing predominantly in the endoplasmic reticulum (ER) of the cancer cells, thus minimizing any undesirable effects involving nuclear DNA.

Typical and atypical domain combinations in human protein kinases: functions, disease causing mutations and conservation in other primates

Ser/Thr and Tyr protein kinases orchestrate many signalling pathways and hence loss in this balance leads to many disease phenotypes. Due to their high abundance, diversity and importance, efforts have been made in the past to classify kinases and annotate their functions at both gross and fine levels. These kinases are conventionally classified into subfamilies based on the sequences of catalytic domains. Usually the domain architecture of a full-length kinase is consistent with the subfamily classification made based on the sequence of kinase domain. Important contributions of modular domains to the overall function of the kinase are well known. Recently occurrence of two kinds of outlier kinases-''Hybrid'' and ``Rogue'' has been reported. These show considerable deviations in their domain architectures from the typical domain architecture known for the classical kinase subfamilies. This article provides an overview of the different subfamilies of human kinases and the role of non-kinase domains in functions and diseases. Importantly this article provides analysis of hybrid and rogue kinases encoded in the human genome and highlights their conservation in closely related primate species. These kinases are examples of elegant rewiring to bring about subtle functional differences compared to canonical variants.

REPERTOIRE OF PROTEIN KINASES ENCODED IN THE GENOME OF ZEBRAFISH SHOWS REMARKABLY LARGE POPULATION OF PIM KINASES

In recent times, zebrafish has garnered lot of popularity as model organism to study human cancers. Despite high evolutionary divergence from humans, zebrafish develops almost all types of human tumors when induced. However, mechanistic details of tumor formation have remained largely unknown. Present study is aimed at analysis of repertoire of kinases in zebrafish proteome to provide insights into various cellular components. Annotation using highly sensitive remote homology detection methods revealed ``substantial expansion'' of Ser/Thr/Tyr kinase family in zebrafish compared to humans, constituting over 3% of proteome. Subsequent classification of kinases into subfamilies revealed presence of large number of CAMK group of kinases, with massive representation of PIM kinases, important for cell cycle regulation and growth. Extensive sequence comparison between human and zebrafish PIM kinases revealed high conservation of functionally important residues with a few organism specific variations. There are about 300 PIM kinases in zebrafish kinome, while human genome codes for only about 500 kinases altogether. PIM kinases have been implicated in various human cancers and are currently being targeted to explore their therapeutic potentials. Hence, in depth analysis of PIM kinases in zebrafish has opened up new avenues of research to verify the model organism status of zebrafish.

Systematic status of Fejervarya ((Amphibia, Anura, Dicroglossidae) from South and SE Asia with the description of a new species from the Western Ghats of Peninsular India

We carried out a large-scale phylogenetic analysis of fejervaryan (dicroglossid frogs with `Fejervaryan lines' on the ventral side of the body) frogs, distributed in South and SE Asia, using published and newly generated sequences of unidentified individuals from the northern Western Ghats. The results corroborate the presence of a larger fejervaryan clade with a sister relationship to a clade composed of Sphaerotheca. Two sister clades could be discerned within the lager fejervaryan clade. The unidentified individuals formed a monophyletic group and showed a strong support for a sister relationship with Minervarya sahyadris. The species was found to be highly divergent (16S rRNA-4% and tyr-1%) from its sister lineage Minervarya sahyadris, and the clade composed of these two lineages were found to be deeply nested within the larger clade of Fejervarya. Based on this, the genus Minervarya Dubois, Ohler and Biju, 2001 is synonymized under the genus Fejervarya Bolkay, 1915. The unidentified lineage is recognized, based on phylogenetic position, genetic divergence and morphological divergence, as a distinct species and named here as Fejervarya gomantaki sp. nov. The presence of rictal glands was observed to be a synapomorphic character shared by the nested clade members, Fejervarya sahyadris and Fejervarya gomantaki sp. nov. Based on the presence of rictal gland and small size, Minervarya chilapata, a species from a lowland region in the Eastern Himalayas, is synonymized under Fejervarya and evidence for morphological separation from the new species, Fejervarya gomantaki sp. nov. is provided. For the fejervaryan frogs, currently three generic names (Frost, 2015) are available for the two phylogenetic subclades; the genus Fejervarya Bolkay, 1915 for the species of fejervaryan frogs having distribution in the South East Asia; the genus Zakerana Howlader, 2011 for the species of fejervaryan frogs having distribution in the South Asia and the genus Minervarya Dubois, Ohler and Biju, 2001 nested within the `Zakerana clade'. In the phylogenetic analysis Minervarya sahyadris, the new species described herein as Fejervarya gomantaki sp. nov. are nested within the `Zakerana clade', if the `Zakerana clade' for the fejervaryan frogs having distribution in the South Asia is provided a generic status the nomen `Minervarya' should be considered as per the principle of priority of the ICZN Code. Taking into consideration the overlapping distribution ranges of members of the sister clades within the larger fejervaryan clade and the absence of distinct morphological characteristics, we also synonymize the genus Zakerana Howlader, 2011, a name assigned to one of the sister clades with members predominantly distributed in South Asia, under the genus Fejervarya Bolkay, 1915. We discuss the need for additional sampling to identify additional taxa and determine the geographical ranges of the members of the sister clades within Fejervarya to resolve taxonomy within this group.

STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis

Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome-melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

水稻富含甘氨酸蛋白在细胞中的定位及其基因调控元件的转基因分析

水稻Angrp-1基因编码富含甘氨酸蛋白质。氨基酸序列分析表明该蛋白在氨基端可能存在一个信号肽序列，其后为富含甘氨酸序列。亲水性指数显示AnGRP-1蛋白包含两个区域。第一个区域由氨基端的1到27氨基酸残基组成。为强疏水性区。该肽段的氨基端为带正电荷号肽特征。与水稻Osgrp-1和菜豆grp1.8基因编码的信号肽进行同源性比较，发现这三种信号肽具有alafLvLLNIg同源序列。AnGRP-1蛋白的第二区域由第28到183氨基酸残基组成。该区域富含甘氨酸，具轻度亲水性，并且含有15个Gly-Tyr-Gly基序，这些基序中的酷西安酸残基之间相互作用，有可能形成分子间或分子内的连接。 利用水稻原生质体瞬间表达系统，比较Angrp-1基因启动子283bp和－1020bp片段，以及Ubiquitin启动子、Actin启动子和35S启动子的活性。结果表明，在水稻原生质体瞬间表达中，Angrp-1基因的两个启动子片段活性较低，接近本底。三个对照的组成型启动子中，以Ubiquitin启动子的活性最强，Actin启动子次之， 35S启动子最弱。根据以上结果推测水稻Angrp-1基因的表达可能具有组织或发育特异异表达和特性。 为研究Angrp-1基因的稳定表达与调控特性，将Angrp-1基因的启动子缺失片段1020bp、941bp、568bp和182bp分别与GUS基因融合，得到pGRP6-121、pGRP7-121、pGRP8-121和pGRP9-121等4个克隆，进行烟草转化，获得转基因植株。GUS活性测定表明在－1020到－568之间，随缺失增多，启动子活性下降。当缺失至－182时，182bp的启动子片段活性高于568bp片段。因此，Angrp-1基因5'端－1020到－568之间存在正调控序列，在－568至－182之间存在负调控序列。对pGRP6-121的烟草转基因植株进行GUS组织染色和活性测发现，GUS基因在维管组织优先表达。在根、茎、叶三种器官中，叶和茎的表达量最高。营养生长期的表达量高于生殖生长期。用2283bp和1020bp的启动子片段分别与GUS基因融合，转化水稻，转化体的GUS组织学染色表明Gus基因具有维管束优先表达特性。因此，Angrp-1基因的表达具有较明显的组织、器官和发育特异性。 水稻AnGRP-1蛋白的免疫组织定位分析表明，Angrp-1基因产物主要定位于水稻的维管组织。利用免疫金方法对AnGRP-1蛋白进行超微结构定位，发现金颗粒主要分布于水稻细胞的细胞壁中。因此，可基本确定AnGRP-1蛋白为细胞壁蛋白。在细胞内的内质网和高尔基体上面或附近也有金颗粒的分布，表明至少有部分AnGRP-1蛋白经过内质网－高尔基体－质膜途径，最终达到细胞壁。 为研究Angrp-1基因编码的信号肽功能，将其信号肽与GUS的氨基端融合，分别置于35S启动子和Angrp-1基因1020bp启动子片段控制之下，得到克隆p35s-SP-GUS-121和pGRP6-SP-GUS-121，分别以pBI121和pGRP6-121作对照，进行烟草基因转化。转化体的GUS活性分析表明，在35S启动子控制下，加与不加信号肽对GUS活性影响不大。但是，在Angrp-1基因的1020bp启动子片段作用下，信号肽与GUS融合后的转化体中其本不表现GUS活性。利用免疫金方法进行的超微结构定位表明，在p35S-SP-GUS-121和pGRP6-SP-GUS-121转化的烟草植株中，均可在细胞壁中检测室GUS产物的存在，因此AnGRP-1的信号肽能引导GUS从细胞质分泌细胞壁中。 以Angrp-1基因5'端的－568到＋1作探针，发现Angrp-1基因以在水稻窄叶青和京系17两个亲本中存在多态性，并进一步将Angrp-1基因定位于水稻第10号染色体上。

光合放氧结构与机理的理论探索

光合放氧研究近十年来已有重要进展，但在该领域仍有很多重要问题待研究。本论文工作主要是对光系统氧化侧与光合放氧密切相关的组分的结构和性能进行理论和模拟研究，工作可概括如下： (一)、放氧中心结构和性能的探索。 1、对放氧中心Mn202单元与配体组氨酸、H2O和Cl等结合方式的理论研究显示：(a)、组氨酸和H20分子均可能与Mn202平面垂直，彼此保持较大的距离，且结合在不同的Mn离子上;(b)、2个H2O分子可能结合在不同Mn202单元上。 2、对Mn簇核心骨架的理论研究显示有必要引入新的Ca、Cl结合方式，以防止因两个Mn202单元线性化而导致2个H2O分子结合位点远离。 (二)、次级电子给体Tyr_z和Tyr_D的结构与功能。 对次级电子给体Tyr_z和Tyr_D进行精确量子化学研究显示：(a)、在中性条件下，Tyr_D和Tyr_z均只与组氨酸通过氢键作用;(b)、当失去电子后，结构发生明显变化，导致正电荷主要集中在组氨酸上，自由基主要集中在Tyr���;(c)、第三组分(H_20分子或羧基等)的引入使中性体系不容易给出电子;(d)、结合最新文献报导，推测Tyr_D~+和Tyr_z~+除与组氨酸作用外还可能分别与水和羧基作用。 (三)、原初电子给体的理论和模拟研究。 1、对紫细菌原初电子给体P_(870)的理论研究显示：(a)、双分子结构比单分子结构稳定;(b)、电荷分离之后，原初电子给体原有的空间结构不再是稳定的构型，它会向能量和化学活性均更低的构型转变。在光合细菌的原初电子给体P870中，这种转变可通过C3位的乙酰基旋转使其氧原子与另一个细菌叶绿素分子的镁原子相互作用使P870+•的总能量和化学活性明显降低。推测这种构型转变对于防止原初反应过程中的电荷重组、维持光能的高效转化有重要意义。提出了原初反应过程中结构动态变化的新观点，利用这一观点可对光合细菌原初反应动力学研究所观测到的慢过程及蛋白质微环境对原初电子给体和原初反应都有重要影响等实验现象给予较好的解释。 2、对光系统II原初电子给体P680的结构进行理论探讨，提出了两个叶绿素a分子平面间夹角为50.0±2.5°时能量最低的夹角模型。 3、采用N-甲基咪唑(C4H6N2)模拟生物体内的组氨酸，通过观测CCL4中的Chla与C4H6N2反应的吸收、CD和MCD光谱得到以下结论：(a)、在纯CCL4中，每个Chla处于5配位状态，Chla形成不对称的双聚体，彼此之间存在较强的偶合作用。提出两个Chla通过不等价的2个Mg-O配位键(O分别来自于C131位的酮基和C17位酯基的C=O)连接为紧密双体结构;(b)、当C4H6N2／Chla = 0.5和1时，其吸收、CD和MCD光谱均发生明显变化，两个Chla之间的偶合作用明显减弱，但此时仍为双聚体。推测C4H6N2首先取代原紧密双体结构中Mg-O酯键，进而取代Mg-O酮键，最后两个Chla分子通过两个Mg…O弱相互作用连接为松弛的双体结构，该模型与理论获得的P_(680)的结构相似。 在上述研究的基础上，提出了包括放氧中心外围配体和TyrZ在内的放氧中心结构新模型。在新结构模型中，2个H2O分子不对称地结合于“C”形结构开口端两个低价的Mn1II和Mn4III上，并保持较大距离;两个组氨酸的咪唑环通过N原子与两个高价的Mn2IV、Mn3IV结合;Cl结合于MB4TM，并与Ca相连;Ca通过O桥和COO-相连使两个Mn202单元保持特定空间构型。TyrZ通过组氨酸(D1-His190)与Mn簇作用。此外，新模型尝试着在O桥上引入质子。放氧中心结构及其邻近环境(包括TyrZ和TyrD)整体处于中性状态。 同时还提出了新的放氧机理，认为电子和质子的释放非同步进行，并首次明确提出两个水分子的不对称氧化和结构动态变化等观点。认为Ca在维持放氧中心的结构方面担负重要作用，C1与Mn离子之间的亲核作用变化是放氧中心结构变化的关键。