干旱胁迫下H_2S信号增强植物叶片的光合作用

干旱胁迫是影响农作物产量最重要的环境因素之一。硫化氢(H_2S)作为第三种气体信号分子在植物体内具有多样且积极的生理功能。目前已了解,H_2S在响应植物干旱胁迫应答以及增强植物光合作用的过程中发挥重要作用,但关于内源性H_2S对干旱胁迫下植物光合作用的调节机制未见报道。该研究以拟南芥哥伦比亚野生型(wild type Col-0,WT)、H_2S产生酶编码基因DES缺失突变体des以及H_2S产生酶编码基因DES过表达突变体OE-DES为实验材料,研究内源性H_2S对干旱胁迫下拟南芥光合作用的调节机制。研究结果显示,植株在正常生长条件下,内源性H_2S促使叶片净光合速率、蒸腾速率、叶绿素含量显著升高;植株遭受干旱胁迫时,内源性H_2S可以显著上调Rubisco和Rubisco活化酶(Rubisco activase,RCA)的表达水平,保护叶绿体结构的完整性,促使叶片净光合速率显著上升,维持叶片相应的蒸腾速率,并且引起叶片气孔关闭和胞间CO_2浓度显著升高。     

硫化氢信号与其它信号交互作用调控植物的耐旱性

硫化氢(H_2S)是继一氧化碳(CO)和一氧化氮(NO)后植物体内发现的第三种气体信号分子,参与种子萌发、植物生长发育及耐逆性的获得等生理过程。干旱是限制作物产量的最主要的环境胁迫因子。近年来,H_2S也已被证实参与植物耐旱性的形成。结合最新的研究进展,在讨论H_2S信号与其它信号分子如活性氧(ROS)、NO、CO、脱落酸(ABA)、乙烯(ETH)、micro RNAs等交互作用的基础上,从气孔运动、渗透调节物质、抗氧化系统、甲基乙二醛脱毒系统、热激蛋白等方面,综述了H_2S诱导植物耐旱性形成的可能机理,并提出了未来的研究方向。进一步拓展了H_2S信号的生理功能和植物耐旱性形成的机理,对深入研究H_2S与植物耐逆性包括耐旱性的关系,具有重要的指导意义。     

肌红蛋白的非酶糖化及其生物学效应

蛋白质的非酶糖化（non－enzymatic glycation）在糖尿病及其并发症的发生发展中起着重要作用。糖化血红蛋白（hemoglobin Alc,HbAlC）已作为糖尿病患者血糖控制的“金标准”。相对而言,同为血红素蛋白的肌红蛋白（myoglobin,Mb）,其非酶糖化的研究相对较少。研究发现：非酶糖化能改变Mb的空间构象,且对Mb血红素活性中心产生一定的影响,从而导致Mb各种生物学功能发生一定程度的改变。本文综述非酶糖化对Mb结构与功能的影响的研究现状,旨在阐明Mb非酶糖化后的结构．性质-功能之间的关系及其生物学意义,为糖尿病及其并发症的发病机制研究提供新的线索。     

细菌血红素降解蛋白结构特点及作用机制

铁离子是几乎所有生物包括细菌生存必需的营养元素. 在宿主体内,绝大多数的铁离子均以血红素的形式存在于各种血红素结合蛋白,如血红蛋白、肌红蛋白等. 当致病菌感染宿主后,血红素将成为某些致病菌主要的铁离子来源. 致病菌编码血红素转运系统,并利用该系统将血红素转运至胞浆,在胞浆血红素被细菌的血红素降解蛋白降解,释放铁离子供细菌利用. 在致病菌中,目前至少有两种血红素降解酶被发现和鉴定. 第一种为经典的血红素氧化酶(heme oxygenase, HO),它催化血红素氧化形成胆绿素、一氧化碳(CO)和Fe2+;第二种非经典降解酶,包括金黄色葡萄球菌的IsdG/IsdI蛋白及其同系物MhuD蛋白,催化血红素分别产生staphylobilin和mycobilin. 另外,部分细菌内存在其它血红素降解因子,其与前两种血红素降解酶无结构同源性,但在血红素降解实验中可产生胆绿素(biliverdin)或CO,因而被鉴定为“血红素降解蛋白”. 对细菌血红素降解蛋白分子结构解析及作用机制的深入理解,将有助于新的血红素降解蛋白的发现和鉴定.     

硫化氢在心血管保护过程中免疫炎症调节研究进展

硫化氢(hydrogen sulfide,H_2S)是一种无色、具有臭鸡蛋气味的气体,过去认为只是一种有毒的气体。近年来大量研究证实H_2S是继一氧化氮(nitric oxide,NO)和一氧化碳(carbon oxide,CO)后第三种内源性气体信号分子,同时,H_2S在心血管系统疾病发生、发展过程中起关键的调控作用,但其机制还不明确,已有报道主要通过抗凋亡、抗氧化、调节内皮一氧化氮合酶活性、促进血管新生等;而本文总结了H_2S在缺血性心脏病、动脉粥样硬化等心血管疾病中免疫炎症调节作用及其机制,从而为H_2S生物学功能以及相关心血管疾病的防治提供新的思路。     

4-硫代胸腺嘧和4-硫胸腺嘧啶脱氧核苷结构的光谱研究

采用拉曼和 UV 光谱研究了抗癌药物4-硫代胸腺嘧和4-硫胸腺嘧啶脱氧核苷的特征结构,确定了药物在酸性、中性和碱性环境下的结构变化。研究结果表明,在酸性和中性溶液中,4-硫代胸腺嘧和4-硫胸腺嘧啶脱氧核苷被强烈质子化,而在碱性环境中则是去质子化,同时,4-硫胸腺嘧啶脱氧核苷的质子化和去质子化比4-硫代胸腺嘧弱,说明氢键的结合对4-硫代胸腺嘧的 NH 质子和溶液分子之间的相互作用具有重要影响。     

4-硫代胸腺嘧和4-硫胸腺嘧啶脱氧核苷结构的光谱研究（英文）

采用拉曼和UV光谱研究了抗癌药物4-硫代胸腺嘧和4-硫胸腺嘧啶脱氧核苷的特征结构,确定了药物在酸性、中性和碱性环境下的结构变化。研究结果表明,在酸性和中性溶液中,4-硫代胸腺嘧和4-硫胸腺嘧啶脱氧核苷被强烈质子化,而在碱性环境中则是去质子化,同时,4-硫胸腺嘧啶脱氧核苷的质子化和去质子化比4-硫代胸腺嘧弱,说明氢键的结合对4-硫代胸腺嘧的NH质子和溶液分子之间的相互作用具有重要影响。     

微生物除臭剂发酵工艺条件优化及硫素转化的研究

利用酵母菌、乳酸菌、醋酸菌三种可食性微生物复配发酵制备微生物除臭剂,研究微生物复配比、发酵时间、发酵温度、接种量四个因素对H_2S去除率的影响。以单因素实验为基础,利用Box-Behnken响应面法优化最佳发酵条件,进一步研究硫元素转化及含量动态变化。结果表明酵母菌、乳酸菌、醋酸菌质量比为1∶2∶2时,各因素对H_2S去除率的影响由高到低依次为发酵温度发酵时间接种量,最优发酵条件为发酵时间48. 5 h、发酵温度30℃、接种量12. 75%,H_2S的去除率可达到71. 84%;实验组与对照组的硫元素转化及含量动态变化相比,实验组的SO_4~(2-)含量显著较高(P0. 05),H_2S释放量显著较低(P0. 05)说明该微生物除臭剂可以调节硫元素转化,有效抑制H_2S产生。     

氢气对辣根过氧化物酶活性的影响及其作用机制的研究

氢气具有广泛的生物学功能,但氢气发挥生物学效应的靶点仍存在争论。我们前期发现,氢气可以与乙酰胆碱酯酶(AChE)直接作用并提高其活性,提示酶分子可能是氢气的潜在作用靶点。除AChE外,氢气是否还可与其它酶分子相互作用目前尚未见报道。本研究目的是探索氢气对辣根过氧化物酶(HRP)活性的影响。采用荧光光谱和紫外光谱方法检测氢气对HRP活性的影响,并对氢分子作用机制进行了深入探索。研究结果表明,当氢气浓度在饱和状态(800μmol/L)和半饱和状态(400μmol/L)下,HRP酶活性分别提高了18.42%和5.44%;荧光光谱检测表明,氢气使HRP内源性氨基酸荧光强度增强了4.15%,色氨酸到血红素中心的距离由2.85 m缩短至2.69 nm,荧光量子产率由72.8%增加至73.4%;紫外光谱检测表明,氢气可使HRP转化为高价铁的HRP I状态速率降低,同时在间歇性紫外照射后,波长在红移至HRP I状态后存在蓝移现象。以上结果提示,氢气可能改变了HRP中的组氨酸、色氨酸及天冬氨酸的微环境,导致氢键网络重构,从而使反应过程中向血红素中心传递能量的效率增加,His42-Asn70之间氢键增强,酶活增强;同时,氢气可使"H_2O-H_2O_2"为桥梁的氢键网络式催化发生变化,降低HRP I的形成速率。本研究不仅进一步证实了酶分子可能是氢分子发挥生物学作用的潜在靶点,同时对氢分子调节HRP酶活性机制的探索,为氢分子作用机制研究提供了更多的线索。     

兼性厌氧细菌硫化氢的产生机理及其生理功能

硫化氢(H_2S)是继一氧化氮(NO)和一氧化碳(CO)后发现的第3种气态信号分子,但其细菌生理学研究才刚刚起步。本文根据作者对奥内达希瓦氏菌的研究,结合新近文献,就细菌的H_2S产生机理及其生理功能作了较为全面的阐述。细菌的H_2S产生途径主要有2条,一是通过降解半胱氨酸产生,二是通过厌氧呼吸产生。产生的H_2S除可为互生性微生物提供能源、供氢体和无机矿质营养外,还具有抑制竞争性微生物的生长,有效占领生态位的作用。H_2S在氧化应答中也起着重要的作用,一方面可抑制过氧化氢酶活性,增加过氧化氢对细菌的杀灭效果;另一方面可作为信号分子激活细菌的氧化应答,诱导拮抗系统的表达,保护细胞免受氧化损伤。这两种看似"矛盾"的作用与H_2S的处理时间有关:短时间处理以抑制为主,而延长处理时间则以保护为主。细菌H_2S产生机理及生理功能的阐明可为硫元素生物地球化学循环规律的揭示和感染性病原细菌的控制提供有益的参考。     

Structural determinants for the formation of sulfhemeprotein complexes

Several hemoglobins were explored by UV-Vis and resonance Raman spectroscopy to define sulfheme complex formation. Evaluation of these proteins upon the reaction with H₂O₂ or O₂ in the presence of H₂S suggest: (a) the formation of the sulfheme derivate requires a HisE7 residue in the heme distal site with an adequate orientation to form an active ternary complex; (b) that the ternary complex intermediate involves the HisE7, the peroxo or ferryl species, and the H₂S molecule. This moiety precedes and triggers the sulfheme formation.     

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O₂) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H₂S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)–SH and heme Fe(II)–O₂ complexes formed by H₂S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O₂ binding site in the heme Fe(II)–O₂ complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H₂S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H₂S combined with H₂O₂ generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H₂S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H₂S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O₂ binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H₂S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.     

Bacterial expression and characterization of molluscan IDO-like myoglobin

The indoleamine 2,3-dioxygenase (IDO)-like myoglobin (Mb) is a unique type of Mb isolated from the buccal mass of several archgastropod species. Here, we expressed Sulculus diversicolor IDO-like Mb as a GST-fusion protein in bacteria. The visible spectrum of GST-fusion IDO-like Mb shows characteristic α- and β-peaks, indicating that it binds oxygen. To identify residues important in heme and oxygen binding, we constructed site-directed mutants. We initially replaced each of the 7 histidines of S. diversicolor IDO-like Mb with alanine. The spectra of three mutants (H74A, H288A, and H332A) revealed a remarkable loss of absorbance around 414 nm, indicating that they cannot bind heme. His⁷⁴, His²⁸⁸, and His³³² were also replaced by arginine or tyrosine. Neither H332R nor H332Y contains heme, suggesting that His³³² is the proximal ligand of IDO-like Mb. In contrast, both H74R and H288Y mutants were isolated in the heme-binding oxy-form. The autoxidation rates of these two mutants showed that they can bind oxygen as stably as wild-type. His⁷⁴ and His²⁸⁸ might be partially associated with heme-binding, but do not act as the distal ligand. The S. diversicolor IDO-like Mb seems to stably bind oxygen in a different manner from normal myoglobins.     

Identification and characterization of VPO1, a new animal heme-containing peroxidase

Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K_m for H₂O₂ of 1.5 mM. When co-expressed in cells, VPO1 can use H₂O₂ produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system.     

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor <Emphasis Type="Italic">Af</Emphasis>GcHK

AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O₂ or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H₂S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O₂, CO, and NO. To address this knowledge gap, we investigated the effects of H₂S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O₂-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na₂S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na₂S, the initially formed Fe(III)–SH heme complex was quickly converted into Fe(II) and Fe(II)–O₂ complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)–SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)–OH complex), whereas in the case of EcDOS the changes in coordination caused by Na₂S treatment led to remarkable increases in catalytic activity.     

Identification and characterization of VPO1, a new animal heme-containing peroxidase

Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K_m for H₂O₂ of 1.5 mM. When co-expressed in cells, VPO1 can use H₂O₂ produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system.     

The multifaceted roles of sulfane sulfur species in cancer-associated processes

Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (H₂S)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that H₂S, currently recognized as a key signaling molecule in human physiology and pathophysiology, plays an important role in cancer biology by modulating cell bioenergetics and contributing to metabolic reprogramming. This is accomplished through functional modulation of target proteins via H₂S binding to heme iron centers or H₂S-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of H₂S but also as key mediators of some of the biological effects commonly attributed to H₂S, the multifaceted role of these species in cancer biology is reviewed here with reference to H₂S, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties.     

Characterization of two cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript> proteins from the cyanobacterium <Emphasis Type="BoldItalic">Gloeobacter violaceus</Emphasis> PCC 7421

In the genome of the untypical cyanobacterium Gloeobacter violaceus PCC 7421 two potential cytochrome b ₆ proteins PetB1 and PetB2 are encoded. Such a situation has not been observed in cyanobacteria, algae and higher plants before, and both proteins are not characterized at all yet. Here, we show that both apo-proteins bind heme with high affinity and the spectroscopic characteristics of both holo-proteins are distinctive for cytochrome b ₆ proteins. However, while in PetB2 one histidine residue, which corresponds to H100 and serves as an axial ligand for heme b _H in PetB1, is mutated, both PetB proteins bind two heme molecules with different midpoint potentials. To recreate the canonical heme b _H binding cavity in PetB2 we introduced a histidine residue at the position corresponding to H100 in PetB1 and subsequently characterized the generated protein variant. The presented data indicate that two bona fide cytochrome b ₆ proteins are encoded in Gloeobacter violaceus. Furthermore, the two petB genes of Gloeobacter violaceus are each organized in an operon together with a petD gene. Potential causes and consequences of the petB and petD gene heterogeneity are discussed.     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Structure-Function Relationships in Unusual Nonvertebrate Globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear.

In this review, special emphasis is placed on the stability properties of the heme-bound O₂. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O₂ to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO₂ or HbO₂ are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.