Biochemical and biophysical studies on cytochrome aa3 II. Conformations of oxidized cytochrome aa3

1.

1. The spectral properties of ‘oxygenated’ cytochrome c oxidase, prepared by passing air through the dithionite-reduced enzyme solution, were compared with those of the ferric enzyme.     

Redox chemistry of low-pH forms of tetrahemic cytochrome c3

Desulfovibrio vulgaris Hildenborough cytochrome c₃ contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c₃ can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV–visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.     

Organometallic chemistry of diphosphazanes. Rhodium(I) complexes of RN(PX2)2 (R = C6H5; X = OC6H5, OC6H4Br−p, R = CH3; X = OC6H5)

Reactions of [Rh(COD)Cl]₂ with the ligand RN(PX₂)₂ (1: R = C₆H₅; X = OC₆H₅) give mono- or disubstituted complexes of the type [Rh₂(COD)Cl₂{ν²−C₆H₅N(P(OC₆H₅)₂)₂}] or [RhCl{ν²−C₆H₅ N(P(OC₆H₅)₂)₂ }]₂ depending on the reaction conditions. Reaction of 1 with [Rh(CO)₂Cl]₂ gives the symmetric binuclear complex, [Rh(CO)Cl{μ−C₆H₅N(P(OC₆H₅)₂)₂} ₂, whereas the same reaction with 2 (R = CH₃; X = OC₆H₅) leads to the formation of an asymmetric complex of the type [Rh(CO)(μ−CO)Cl{μ−CH₃N(P(OC₆H₅)₂)₂}₂ containing both terminal and bridging CO groups. Interestingly the reaction of 3 (R = C₆H₅, X = OC₆H₄Br−p with either [Rh(COD)Cl]₂ or [Rh(CO)₂Cl]₂ leads only to the formation of the chlorine bridged binuclear complex, [RhCl{ν²−C₆H₅N(P(OC₆H₄Br−p)₂)₂}]₂. The structural elucidation of the complexes was carried out by elemental analyses, IR and ³¹P NMR spectroscopic data.     

The respiratory chain of plant mitochondria. XV. Equilibration of cytochromes c549, b553, b557 and ubiquinone in mung bean mitochondria: Placement of cytochrome b557 and estimation of the midpoint potential of ubiquinone

1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b₅₅₇, b₅₅₃, c₅₄₉ (corresponding to c₁ in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis.

2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c₅₄₉, b₅₅₇, and b₅₅₃. The intramitochondrial redox potential (IMP_h) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition.

3. 3. This result shows that the slow reduction of cytochrome b₅₅₇ under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b₅₅₃ and +235 mV for cytochrome c₅₄₉. Cytochrome b₅₅₇ is placed on the low potential side of coupling site II and transfers electrons to cytochrome c₅₄₉ via the coupling site.

4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMP_h with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMP_h was calculated. When replotted as IMP_h versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential E_m7.2 = + 70 mV. The minor component has a midpoint potential E_m7.2 = − 12 mV; its nature is unknown.

转CBF_1基因棉花的耐旱生理研究

以转CBF_1基因棉花及其野生型棉花为材料,设置轻度(900 mL)、中度(400 mL)、重度(300 mL)和对照(1 200mL)浇水处理的不同干旱胁迫和复水处理,考察各种处理后对盆栽植株不同部位叶片光合性能和离体叶片在暗处理条件下叶绿素含量的变化,以及在大田苗期、蕾期、花期、铃期断水胁迫对棉花产量的影响,为转基因抗旱棉花新品种的培育提供理论依据。结果表明:(1)在盆栽试验中,随着干旱胁迫时间的延长和复水3d处理,各浇水处理的转基因和野生型棉花叶片净光合速率(P_n)、原初光能转化效率(F_v/F_m)表现出先降低后增加的变化趋势,受到胁迫后对顶部叶片的影响比中部叶片大,且转基因棉花叶片保留数量显著高于野生型棉花;它们的离体叶片叶绿素a、叶绿素b的含量随着干旱胁迫时间的延长而逐渐下降,但转基因棉花的下降速率显著低于野生型棉花。(2)在田间试验的苗期、蕾期、花期、铃期干旱胁迫下,各干旱胁迫处理的转基因植株的皮棉产量、衣分、种子质量、株高均显著高于野生型棉花;转基因棉花的籽棉产量分别比正常灌溉处理降低了78.4%、55.1%、12.7%、8.3%,野生型棉花则分别降低了80.4%、55.4%、19.2%、14.4%,不同时期的水分胁迫严重影响了棉花籽棉产量,但是转基因棉花的籽棉产量显著高于野生型棉花。研究认为,在不同干旱胁迫条件下,转CBF_1基因棉花表现出优良的生长和生理优势,可提高棉花的耐旱性。     

黄花棘豆脱水素OoY_2K_4的鉴定及表达分析

该研究以黄花棘豆cDNA为模板,采用同源克隆法,从黄花棘豆转录组数据库中克隆获得1个响应逆境胁迫的胚胎发育晚期丰富蛋白基因,命名为OoY_2K_4;OoY_2K_4基因ORF为786bp,编码261个氨基酸,含有2个保守的Y片段和4个K片段,为典型的Y_2K_4类脱水蛋白亚家族成员;OoY_2K_4蛋白不具有跨膜结构域,不存在信号肽,亲水性极强,含有1个糖基化位点和17个磷酸化位点;亚细胞定位显示,OoY_2K_4蛋白定位于细胞质中。多序列比对发现,OoY_2K_4蛋白与其他物种第二组LEA蛋白(脱水素)序列高度保守;进化树分析显示,该序列与三叶草、蒺藜苜蓿和紫花苜蓿相似度最高,亲缘关系最近。采用qRT-PCR对OoY_2K_4基因在干旱、高盐、低温以及脱落酸、乙烯、赤霉素处理下的表达分析显示,干旱和高盐胁迫可显著诱导OoY_2K_4基因表达,而低温胁迫下基本无变化;激素处理均可诱导OoY_2K_4基因高效表达,其中脱落酸诱导下OoY_2K_4基因表达最显著。研究推测,OoY_2K_4基因可能通过依赖ABA的信号途径参与黄花棘豆对干旱和高盐逆境胁迫的应答反应。     

Haem—haem interactions in cytochrome aa3 during the anaerobic-aerobic transition

A biphasic response is seen at both 445 and 605 nm as the ascorbate—cytochrome c—cytochrome aa₃ system is taken slowly from the anaerobic to the aerobic state. At low oxygen tensions the 445 nm band is more reduced while at high oxygen tensions the 605 nm band is more reduced. It is suggested that the redox potential for cytochrome a (contributing 70% at 605–630 nm and 50% at 445–455 nm) is a function of the redox state of cytochrome a₃. This model can account for both the aerobic/anaerobic data and for observations of interactions in the anaerobic system alone (Leigh, Jr, J.S., Wilson, D.F., Owen, C.S. and King, T.E. (1974) Arch. Biochem. Biophys. 160, 476–486).     

A regulatory system controlling the production of cytochrome aa3 in Neurospora crassa

Summary The mitochondria of the cyt-2-1, cya-3-16, cya-4-23 and 299-1 nuclear mutants and the [mi-3] and [exn-5] cytoplasmic mutants of Neurospora crassa are deficient in cytochrome aa ₃, while the cyb-1-1 and cyb-2-1 mutants have mitochondrial b-cytochrome dificiencies. However, the mitochondria from cyb-1-1 cyt-2-1, cyb-1-1 [mi-3] and cyb-2-1 [mi-3] double mutants contain 30% to 50% of the amount of cytochrome aa ₃ that is present in mitochondria from wild-type; i.e. cyb-1-1 and cyb-2-2 act as suppressors of the cytochrome aa ₃ deficiency phenotypes that are associated with the cyt-2-1 and [mi-3] mutations.The production of cytochrome aa ₃ can be induced in cyt-2-1 and [mi-3] by growing cells in medium containing antimycin A, an inhibitor of electron transport in the cytochrome bc ₁ segment of the mitochondrial electrontransport chain. Moreover, the growth of the [mi-3] mutant is strongly stimulated by low concentrations of antimycin A. The induction of cytochrome aa ₃ by antimycin treatments does not occur in [exn-5], cya-4-23 and 299-1 cells, but does take place in cya-3-16 cells.Although some of the seven constituent polypeptides of cytochrome aa ₃ are present the mitochondria of [mi-3], the holoenzyme complex is not formed in the mutant. In contrast, the mitochondria of cyb-1-1 [mi-3] and cyb-2-2 [mi-3] double mutants contain a fully assembled cytochrome oxidase complex as well as some unassembled subunit polypeptides.The observations are indicative of the existence of at least two regulatory systems controlling the production of cytochrome aa ₃. One of the circuits appears to control the basal or constitutive production of cytochrome oxidase, the other seems to coordinate the level of cytochrome aa ₃ with some function of the mitochondrial cytochrome bc ₁ complex, possibly electron transport.     

百子莲脱水素基因ApY2SK2逆境应答模式及启动子调控功能研究

在百子莲胚性细胞中筛选到对超低温保存复合逆境具有积极响应的保护类蛋白脱水素(ApY_2SK_2),为探明ApY_2SK_2基因在复合逆境中的应答模式,该研究采用染色体步移技术克隆并分析了ApY_2SK_2编码基因上游1 200 bp的启动子序列。结果表明:(1)序列分析显示,该启动子含有多个与逆境和激素诱导相关的顺式调控元件;实时荧光定量PCR结果表明,ApY_2SK_2基因的表达具有组织特异性,在百子莲的叶和果中表达量较高,且在多种胁迫处理与ABA激素诱导下,其表达量显著升高。(2)成功构建了5个ApY_2SK_2启动子不同缺失片段驱动GUS基因的融合表达载体,经农杆菌转化、抗性筛选和PCR检测鉴定,获得T_3代纯和转基因拟南芥株系。(3) GUS组织化学染色结果显示,GUS基因在拟南芥幼苗全株、成年苗的叶、花和成熟果实中表达活性较强,但在未成熟果实中无明显表达;烟草瞬时表达结果显示,与对照组相比,在脱水胁迫和ABA处理下的ApY_2SK_2启动子不同缺失片段驱动GUS基因表达具有显著差异。(4)转基因拟南芥GUS活性测定结果显示,ApY_2SK_2启动子MBS元件和ABRE元件可响应干旱与渗透胁迫信号;ApY_2SK_2启动子LTR元件参与低温响应;ApY_2SK_2启动子-1 199～-262 bp区域包含多个串联的ABRE顺式调控元件(-373～-211 bp)对响应ABA信号具有主要调控作用。该研究结果揭示了ApY_2SK_2启动子的组织特异性,且启动子上的关键顺式调控元件对不同的胁迫和激素信号响应具有决定性调控作用。     

Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [d-Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.     

枸杞WRKY_3基因克隆及组织表达分析

以枸杞为材料,采用PCR及RACE方法,克隆了枸杞WRKY转录因子基因cDNA序列,命名为Lb WRKY3,GenBank登录号为KX196192。在生物信息学分析的基础上,进行亚细胞定位、基因表达分析。结果显示:(1)Lb WRKY3开放阅读框ORF长度为1 068bp,编码356个氨基酸。(2)生物信息学分析显示,Lb WRKY3编码蛋白具有一个WRKY结构域,二级结构中不规则卷曲结构所占比例最大(58.67%),延伸链结构次之(18.88%),α螺旋比例为15.82%,β转角最少,仅为6.63%;Lb WRKY3蛋白与案头菊WRKY蛋白、黄花蒿WRKY蛋白相似性较高。(3)亚细胞定位显示,Lb WRKY3蛋白定位于细胞核。(4)实时定量PCR分析表明,Lb WRKY3在根中表达量最高,在花中表达量最低;在枸杞果实发育过程中Lb WRKY3均有表达,表达量随果实成熟逐渐升高,并于35d达到峰值;Lb WRKY3基因在果实中的表达具有组织特异性表达特性(果肉果皮种子)。研究表明,Lb WRKY3基因参与了枸杞果实生长发育调控。     

转天山雪莲sikPIP3基因烟草的获得及抗逆性鉴定

利用PCR技术从实验室建立的天山雪莲DNA文库中克隆了天山雪莲质膜水孔蛋白基因sikPIP3,构建了植物表达载体pBI121-sikPIP3,通过农杆菌介导法转化烟草品种NC89,经PCR和RT-PCR检测证明目的基因成功导入并得到了表达,采用水分胁迫进行抗旱分析和采用冷冻胁迫进行抗寒性分析。结果显示:(1)克隆出具有水孔蛋白基因特性的sikPIP3基因。(2)经断水干旱处理,转基因烟草的生长表型优于野生型烟草,特别是在断水9d的情况下,野生型烟草已经完全萎蔫,转基因烟草萎蔫症状较轻;生理指标测量结果显示,转基因烟草的相对电导率和MDA的含量低于野生型烟草,相对含水量和CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗旱性高于野生型烟草。(3)经不同温度胁迫处理,转基因烟草的生长表型优于野生型烟草,特别是在-4℃冷处理6h情况下,野生型烟草已经完全萎蔫,转基因烟草只出现少量伤斑;生理指标分析结果表明,转基因烟草的相对电导率和MDA的含量低于野生型烟草,CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗寒性高于野生型烟草。综合结果表明:sikPIP3基因在抗逆基因工程方面具有较高的应用前景。     

Cytochrome c551 as a model system for protein folding

Considerable progress was made over the last few years in understanding the mechanism of folding of cytochrome c₅₅₁, a small acidic hemeprotein from Pseudomonas aeruginosa. Comparison of our results with those obtained by others on horse heart cytochrome c allows to draw some general conclusions on the structural features that are common determinants in the folding of members of the cytochrome c family.     

Chloroplast cytochrome b6: Molecular composition as a lipoprotein

Disc electrophoretically homogeneous spinach-chloroplast cytochrome b₆ was found to be a lipoprotein whose redox potential was essentially unchanged during isolation. These results further support the hypothesis of Triton X-100/4 M urea, pH 8, as a useful extracting medium for membrane lipoproteins.

Cytochrome b₆ was found to have a heme equivalent dry weight of 1 mol of heme per 60 000 g. Of this, 20 000 g was lipid-extractable. The molecular weight was 60 000 with a partial specific volume of 0.84 ml/g. The protein portion of the molecule (40 000) consisted of 1 polypeptide chain of 20 000 daltons, 1 of 9600 daltons and 2 of 6600 daltons. A simple lipid composition (relative to the original membrane) was found consisting of 7 mol of chlorophyll a and 6 mol of cardiolipin per mol of cytochrome; these two lipids thus account for about 75–80% of the lipid content. An unidentified minor neutral lipid and minor polar lipid were also detected. At pH 7.0 in the presence of 0.5% Triton X–100, E′₀ was −0.080 V, and in the absence of Triton X–100, E′₀ was −0.120 V. At pH 8 in 0.5% Triton X–100, E′₀ was −0.084 V, thus indicating that the redox potential is independent of pH in the region 7–8. The redox reaction proceeded via a one-electron-transfer.     

Solution-mediated syntheses and characterizations of two new zincophosphites [C6H14N2]0.5[Zn(H2PO3)2] and [C4H12N2]0.5[(CH3)2NH2][Zn2(HPO3)3]

Two new zincophosphites [C₆H₁₄N₂]_0.5[Zn(H₂PO₃)₂] 1 and [C₄H₁₂N₂]_0.5[(CH₃)₂NH₂][Zn₂(HPO₃)₃] 2 have been solvothermally synthesized in mixed solvents of N,N-dimethylformamide (DMF) and 1,4-dioxane (DOA), respectively. Single-crystal X-ray diffraction analysis reveals that compound 1 exhibits a neutral inorganic chain formed by ZnO₄ and HPO₂(OH) units. Interestingly, the left- and right-handed hydrogen-bonded helical chains are alternately formed via the hydrogen-bonds between two adjacent chains. Compound 2 exhibits a layer structure with 4- and 12-MRs formed by ZnO₄ and HPO₃ units, in which two kinds of organic amine molecules both act as countercations to compensate the overall negative electrostatic charge of the anionic network.