竹红菌甲素与细胞色素C相互作用的同步荧光光谱研究

利用同步荧光光谱方法研究了在生理pH值条件下竹红菌甲素（HA）对细胞色素C分子构象的影响，同时探讨其作用机理．同步荧光光谱实验结果说明HA与细胞色素C之间的相互作用主要发生在HA和细胞色素C分子表面的氨基酸残基之间，最终这种作用使细胞色素C的构象发生变化．     

细胞色素C在汞表面生长性质的AFM研究——吸附自组装机理

利用原子力显微镜对细胞色素C在汞表面生长过程进行研究,发现细胞色素C在汞表面首先形成的是高度稍大于1nm的吸附单分子层,随着生长时间的增加,吸附在汞表面的细胞色素C分子和溶液中的分子问相互作用,自组装形成生长活性点,生长速度明显加快,生长时间达到8min时,细胞色素C能形成底部直径达527nm,高度达138nm,分布密度约为1．2～2．3个/μm^2的细胞色素C分子集聚体．生长机理是吸附自组装机理．细胞色素C的浓度较大地影响其在Hg表面的自组装平衡,随着浓度的减少,细胞色素C所能形成的最大分子集聚体的高度和数目都随之减少。浓度与平均表现高度是非线性的关系．     

细胞色素C的分子静电势研究

本文利用自制的计算机程序,计算了金枪鱼亚铁细胞色素C的分子静电势,并据此讨论了细胞色素C的结构和性能,得出了一些有意义的结果.     

猪心细胞色素C对固氮酶的作用

在我们早期的工作中,发现棕色固氮菌无细胞抽提物经过柱分离,得到单独没有固氮活性的钼铁蛋白部分,如果加入适当量的猪心细胞色素C时,就能表现出还原分子氮和乙炔的活性。后来的工作表明,当镏铁蛋白组分纯度提高以后,细胞色素C的作用不但没有提高,反而下降了。因此,我们对细胞色素C的作用做了进一步的分析研究。     

细胞色素C在电沉积PAMAM—HA修饰电极上的电化学行为

采用双电位阶跃法在铂电极上电沉积制备聚酰胺胺一羟基磷灰石复合涂层，研究了细胞色素C在复合涂层修饰的铂电极上的电化学行为，循环伏安测量结果表明，细胞色素C在修饰电极上呈现一对氧化还原电流峰，表现为扩散控制的准可逆的电子转移过程、重点探讨了涂层的厚度对促进细胞色素C电子传递的影响．对电化学交流阻抗谱的测试结果进行了相关的电化学参数的拟合，讨论了该修饰电极促进细胞色素C电子传递可能的机制．     

细胞色素C在葡聚糖修饰金电极上的直接电化学

本文对细胞色素C在葡聚糖修饰金电极上的直接电化学进行了研究,发现萄聚糖是细胞色素C电化学反应的有效促进剂,并对影响萄聚糖修饰金电极的稳定性的因素作了探讨.     

细胞色素C修饰电极的制备和性能研究

通过自组装法,把血红蛋白细胞色素C固定于导电聚合物(聚谷氨酸)层,制作成一种微型生物传感器,检测其对过氧化氢和亚硝酸钠的响应.实验发现谷氨酸具有一定的电化学活性,循环伏安图在-0.35 V和-0.05 V出现一对氧化还原峰,而且细胞色素C修饰电极对亚硝酸钠响应所得循环伏安图表明,随着亚硝酸钠加入量增大,还原峰增大.通过吸附法修饰电极,把血红蛋白细胞色素C固定于电极表面,探究修饰电极在对苯二胺中的钝化情况.实验表明:细胞色素C修饰电极可以避免电极钝化的发生.细胞色素C作为一种蛋白酶,使对苯二胺处于氧化状态,从而防止电极表面膜的形成,避免电极钝化的发生.     

固氮酶固氮机理研究——Ⅰ.猪心细胞色素C与钼铁蛋白重组合

以猪心细胞色素C代替棕色固氮菌铁蛋白,与棕色固氮菌钼铁蛋白重组合,实现了氮还原成氨,乙炔还原成乙烯,以及需还原剂的ATP水解反应。猪心细胞色素C与棕色固氮菌钼铁蛋白重组合亦要求一定的比例。从差示光谱上看,猪心细胞色素C与棕色固氮菌铁蛋白在波长460mμ处都有一个共同的吸收峰。实验结果表明,钼铁蛋白本身可能就是固氮酶;而铁蛋白起着电子传递体的作用。     

细胞色素C/L-半胱氨酸修饰电极的电化学行为研究

采用自组装修饰法将细胞色素C修饰到以L-半胱氨酸为连接剂的金电极上,并运用循环伏安法(CV)、电化学阻抗法(EIS)等方法研究了该电极的电化学行为.测定了细胞色素C有关的电化学参数.     

乙醇对细胞色素c和脱辅基细胞色素c的结构转换影响

用紫外－可见吸收光谱,荧光光谱和圆二色谱对细胞色素c和脱辅基细胞色素c在不同的φ（乙醇）溶液中的结构进行了表征。结果表明,乙醇对细胞色素c的α－螺旋二级结构影响很小,而对血红素配位的影响十分明显,可导致血红素Fe-Met80配位键断裂,另一方面,乙醇对脱辅基细胞色素c有明显的结构转换诱导作用,在φ（C2H5OH）＝0．55（乙醇／水）溶液中,脱辅基细胞色素c的α－螺旋结构含量可以达到全蛋白的89％,研究结果表明：负电荷微环境的诱导对脱辅基细胞色素c的结构转换并不是必要的,改变溶剂与脱辅基细胞色素c之间的疏水和氢键相互作用力也可以诱导脱辅基细胞色素cα-螺旋的形成。     

Bis-methionine axial ligation of haem in bacterioferritin from Pseudomonas aeruginosa

The iron-containing bacterioferritins contain the protoporphyrin IX haem group. It has been established that Escherichia coli cytochrome b1, cytochrome b557 and bacterioferritin are identical. The optical spectra at room temperature of the haem group show it to be predominantly low-spin in both the ferrous and ferric states. The nature of the axial ligands binding the haem group to the polypeptide has, however, remained unknown. Low-spin, bis-coordinate haem centres in proteins typically have a role in rapid electron transfer as redox changes at the metal ion lead to little structural rearrangement. There are only four amino acids with side-chains that have ligand field strengths sufficient to generate the low-spin state of haem, namely, histidine, lysine, methionine and cysteine. Hence there are, potentially, ten different pairs of these four ligands which could be discovered in electron transfer haemoproteins. To date only three have been established with certainty. They are bis-histidine, as in mammalian cytochrome b5, methionine-histidine, typified by cytochrome c and lysine-histidine, recently recognized by spectroscopic methods in cytochrome f. Here we report the electron paramagnetic resonance and near infrared magnetic circular dichroism spectra of the oxidized state of Ps. aeruginosa bacterioferritin which enable the axial ligands to be identified as the thioether side chains of two methionine residues, a ligation scheme not previously reported for haem in any protein.     

Functional relationship of cytochrome c(6) and plastocyanin in Arabidopsis

Photosynthetic electron carriers are important in converting light energy into chemical energy in green plants. Although protein components in the electron transport chain are largely conserved among plants, algae and prokaryotes, there is thought to be a major difference concerning a soluble protein in the thylakoid lumen. In cyanobacteria and eukaryotic algae, both plastocyanin and cytochrome c(6) mediate electron transfer from cytochrome b(6)f complex to photosystem I. In contrast, only plastocyanin has been found to play the same role in higher plants. It is widely accepted that cytochrome c(6) has been evolutionarily eliminated from higher-plant chloroplasts. Here we report characterization of a cytochrome c(6)-like protein from Arabidopsis (referred to as Atc6). Atc6 is a functional cytochrome c localized in the thylakoid lumen. Electron transport reconstruction assay showed that Atc6 replaced plastocyanin in the photosynthetic electron transport process. Genetic analysis demonstrated that neither plastocyanin nor Atc6 was absolutely essential for Arabidopsis growth and development. However, plants lacking both plastocyanin and Atc6 did not survive.     

Site-directed mutagenesis of cytochrome c shows that an invariant Phe is not essential for function

Phenylalanine 87 of yeast iso-1-cytochrome c (Phe 82 in horse heart and bonito) is phylogenetically conserved and occurs near the surface of the protein. It has been suggested that this residue is directly involved in electron transfer between cytochrome c and cytochrome c peroxidase (CCP) and may also control the polarity of the haem environment. Because Phe residues are not susceptible to chemical modification, no direct means of studying the functional role of Phe 87 has been available, so we have chosen Phe 87 as our initial target here to test the feasibility of using site-directed mutagenesis as a means of studying structure-function relationships in cytochrome c. We have changed the codon for Phe 87 to that of either a Ser, a Tyr or a Gly. The mutated genes have been introduced into a yeast strain lacking both isozymes of cytochrome c. Unlike the recipient strain, transformants grow on a non-fermentable carbon source, indicating that the mutant proteins can reduce cytochrome oxidase. The purified mutant proteins are similar to wild type with respect to their visible spectra, 20-70% as active as wild-type protein in the CCP assay, and their reduction potentials are lowered by as much as 50 mV. Thus Phe 87 is not essential for cytochrome c to transfer electrons but is involved in determining the reduction potential.     

The X-linked chronic granulomatous disease gene codes for the beta-chain of cytochrome b-245

Chronic granulomatous disease (CGD) is a rare inherited disorder associated with a profound predisposition to infection due to the lack of a microbicidal oxidase system in the phagocytes of these patients. This syndrome is most commonly inherited through a defect on the X chromosome and the only clearly defined component of the oxidase system, the very unusual cytochrome b (b-245), has been shown to be missing from the cells of these patients. This cytochrome is a heterodimer composed of an alpha-chain of relative molecular mass (Mr) 23,000 (23K) and a 76-92K beta-chain; neither are detectable in neutrophils from X-linked CGD subjects. The defective X-CGD gene has recently been cloned by 'reverse genetics' but the protein predicted from the proposed complementary DNA sequence was not identified. We have purified the beta-chain of the cytochrome and sequenced 43 amino acids from the N terminus. Almost complete homology was obtained between this sequence and that of the complementary nucleotides 19-147 of the sequence of the X-CGD gene, originally designated as a non-coding region.     

猪心肌细胞色素C两种纯度测定法的比较和结晶的制备

本文介绍了用纯化的猪心肌细胞色素C—与溶菌酶掺和的样品,比较其百分含铁量法和E_(280)~(c2+)/E_(280)~(c3+)比值法两种不同的纯度测定方法,证明二者有很好的线性关系。光谱比值法与百分铁量法比较,有需要样品少,操作简便、迅速等优点。 此外还报导了经纯化和浓缩的猪心肌细胞色素C用饱和硫铵制备出结晶的简便方     

Identification of the BAL-labile factor

One of us has previously reported that treatment of the Keilin and Hartree heart-muscle preparation with 2,3-dimercaptopropanol (BAL), in the presence of air, leads to the complete inactivation of the succinate oxidase system with little if any effect on the activities of succinate dehydrogenase (until more than half the BAL was oxidized) or cytochrome c oxidase. The inactivation of the complete succinate oxidase system requires the oxidation of BAL by air in the presence of the enzyme. It is not caused by H2O2 or BAL disulphides produced during the oxidation of BAL. Spectroscopic studies identified the block as lying between cytochromes b and c. It was suggested that a BAL-labile factor is present which transfers electrons from cytochrome b to cytochrome c and which is destroyed by coupled oxidation with BAL. The factor is also required for NADH oxidation. Subsequent work showed it is not identical with cytochrome c1 (ref. 4), myoglobin present in the preparation or the antimycin-binding site. We report here that this factor is identical to the iron-sulphur protein in the central portion of the respiratory chain first identified by Rieske.     

纳米火棉胶膜自组装细胞色素c的直接电化学研究

用纳米火棉胶膜将细胞色素c固定在玻碳电极表面,制备了细胞色素c-火棉胶膜修饰电极.吸附在火棉胶膜上的细胞色素c可以与电极发生直接电子传递.在pH=7.0的0.1mol/LPBS缓冲溶液中可得到一对准可逆的细胞色素c的血红素辅基Fe(Ⅲ)/Fe(Ⅱ)电对氧化还原峰,实验求得细胞色素c异相电子传递速率常数k0为65.4μm/s.进一步考察了扫速、溶液pH值等因素对细胞色素c电子传递的影响,并用电化学阻抗法研究了修饰电极的电化学行为.     

Structural insights into electron transfer in caa3-type cytochrome oxidase

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme's function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36?? resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain, which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.     

K_2PtCl_4与细胞色素C修饰体系研究

马心细胞色素C(以下简写为Cyt·C)与K_2PtCl_4的反应混合物在Sephadex G-75上分出4个组分,对应于Cyt·C寡聚体、三聚体、二聚体及单体,此结果与SDS聚丙烯酰胺凝胶电泳(以下简写为SDS-PAGE)测定的各组分的分子量相一致,这揭示了K_2PtCl_4与Cyt·C存在多位点修饰,而非如Kostic报道的Met-65为其唯一修饰位点,此结论通过分析K_2PtCl_4与His-33羧甲基化的Cyt·C修饰反应产物所证实,同时也和同样条件下游离His,Met与K_2PtCl_4的配位作用相吻合,对Cyt·C二聚体衍生物的还原电位、紫外-可见光谱以及二阶导数傅利叶变换红外光谱研究表明,血红素周围的电子环境仍然保持完整,但其二级结构有较大的扰动.     

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b 5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b 5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b 5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b 5 with cytochrome c are: wild type K A=4.2(±0.01)×10 6(mol/L) -1 , Phe35Tyr K A=3.7(±0.01)×10 6(mol/L) -1 and Phe35Leu K A=4.7(±0.01)×10 6(mol/L) -1 respectively ( I =1 m mol/L, pH 7.0 soldium phosphate buffer, 25℃). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b 5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b 5 and cytochrome c.