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结核分枝杆菌铁氧还蛋白还原酶FdrA和FprA在CYP125A1的电子传递链中的作用分析

中国医药生物技术 2012年第3期7卷 178-186页

作者：乔峰张健美白银磊杨信怡李聪然李国庆胡辛欣游雪甫中国医学科学院北京协和医学院医药生物技术研究所药理室北京100050

目的体外评价结核分枝杆菌(Mtb)铁氧还蛋白还原酶FdrA和FprA的活性,探索它们分别与两种铁氧还蛋白的偶联作用,并分析它们在CYP125A1的电子传递链中的作用。方法采用大肠杆菌作为宿主克隆结核分枝杆菌FdrA、FprA和CYP125A1编码基因并进... 详细信息

目的体外评价结核分枝杆菌(Mtb)铁氧还蛋白还原酶FdrA和FprA的活性,探索它们分别与两种铁氧还蛋白的偶联作用,并分析它们在CYP125A1的电子传递链中的作用。方法采用大肠杆菌作为宿主克隆结核分枝杆菌FdrA、FprA和CYP125A1编码基因并进行蛋白外源表达;以NADH或NADPH为电子供体,2,6-二氯酚靛酚(DCPIP)为电子受体评价FdrA及FprA的活性;应用细胞色素C为电子受体研究FdrA或FprA与不同铁氧还蛋白的偶联作用;分析CYP125A1对4-胆甾烯-3-酮的代谢作用进而研究FdrA和FprA在CYP125A1的电子传递链中的作用。结果 FdrA对NADH亲和力较高,Fdx对FdrA活性有明显提升作用,菠菜铁氧还蛋白(spFDX)对其活性没有提升作用,FdrA/Fdx和FdrA/spFDX均不能支持CYP125A1的活性。FprA对NAPDH亲和力较高,Fdx和spFDX均对FprA活性有明显提升作用,Fdx尤甚,FprA/spFDX可以支持CYP125A1的活性,FprA/Fdx不能支持CYP125A1的活性。结论 FprA是结核分枝杆菌CYP125A1的电子传递链蛋白,FdrA可能不是CYP125A1的电子传递链蛋白。

关键词：分枝杆菌结核铁氧还蛋白NAPP还原酶细胞色素P450酶系统电子传递链复合蛋白质类

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Regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii by CydR (Fnr) -: Sensitivity to oxygen, reactive oxygen species, and nitric oxide

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第7期275卷 4679-4686页

作者： Wu, GH Cruz-Ramos, H Hill, S Green, J Sawers, G Poole, RK Univ Sheffield Krebs Inst Biomolec Res Dept Mol Biol & Biotechnol Sheffield S10 2TN S Yorkshire England John Innes Ctr Plant Sci Res Nitrogen Fixat Lab Norwich NR4 7UH Norfolk England

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant nitrogen fixation requires cytochrome bd, Regulation of cytochrome bd expression is achieved by CydR (an Fnr homologue), which represses transcription of the oxidase genes cydAB. cydAB mRNA was mapped by primer extension;the transcriptional start site was determined, and putative -10 and -35 regions were deduced. Two "CydR boxes," one at the +1 site and one upstream of the -35 region, were identified. Transcriptionally inactive, purified CydR was converted, by adding NifS, cysteine, and Fe2+, into an active form possessing acid-labile sulfide and spectra suggesting a [4Fe-4S](2+) cluster. Reconstituted CydR specifically bound both CydR boxes cooperatively, with higher affinity for the nearer consensus +1 site. Low concentrations of O-2 or NO ([O-2]/[CydR] or [NO]/[CydR] = 0.1-0.6) elicited loss of the 420 nm absorbance attributed to the [4Fe-4S](2+) cluster, formation of a 315 nm species, and loss of ability to retard DNA migration. Retardation by reconstituted CydR was enhanced by superoxide dismutase and/or catalase, suggesting a role for reactive oxygen species in CydR inactivation. The role of CydR in regulating cydAB expression in the supposedly anoxic cytoplasm of A, vinelandii and similarities to cydAB regulation by Fnr in Escherichia coli are discussed.

关键词：维涅兰德固氮菌/酶学细菌蛋白质类碱基序列细胞色素类/遗传学 DNA引物电子传递链复合蛋白质类大肠杆菌蛋白质类基因表达调控细菌/生理学基因表达调控酶学/生理学诱变定点一氧化氮/代谢氧化还原酶类/遗传学氧/代谢活性氧/代谢阻遏蛋白质类/遗传学阻遏蛋白质类/分离和提纯阻遏蛋白质类/生理学转录遗传

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Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd:: A di-heme active site?

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第4期97卷 1554-1559页

作者： Vos, MH Borisov, VB Liebl, U Martin, JL Konstantinov, AA Ecole Natl Super Tech Avancees Ecole Polytech Lab Opt Appl INSERMU451 F-91761 Palaiseau France Moscow MV Lomonosov State Univ AN Belozersky Inst Physicochem Biol Moscow 119899 Russia

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part: of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

关键词：结合部位一氧化碳/化学细胞色素a组/化学细胞色素类/化学细胞色素a1类电子传递链复合蛋白质类大肠杆菌/酶学大肠杆菌蛋白质类血红素/类似物和衍生物血红素/化学血红素/代谢动力学配体氧化还原氧化还原酶类/化学分光光度法

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A cytochrome bb′-type quinol oxidase in Bacillus subtilis strain 168

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第46期274卷 32810-32817页

作者： Azarkina, N Siletsky, S Borisov, V von Wachenfeldt, C Hederstedt, L Konstantinov, AA Lund Univ Dept Microbiol Solvegatan 12 SE-22362 Lund Sweden Moscow MV Lomonosov State Univ AN Belozersky Inst Physicochem Biol Moscow 119899 Russia

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa, and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase, The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component;however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases;in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase super-family. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cfa and qox are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively, Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.

关键词：枯草芽孢杆菌/酶学枯草芽孢杆菌/遗传学一氧化碳/药理学细胞呼吸细胞色素类/化学细胞色素类/遗传学细胞色素类/代谢电子传递链复合蛋白质类电子传递复合物Ⅳ/化学电子传递复合物Ⅳ/遗传学酶抑制剂/药理学大肠杆菌蛋白质类基因细菌葡萄糖/药理学血红素/分析膜蛋白质类/代谢多酶复合物/拮抗剂和抑制剂突变 NADH NADPH氧化还原酶类/拮抗剂和抑制剂氧化还原酶类/遗传学氧化还原酶类/代谢质子喹诺酮类/药理学氰化钠/药理学分光光度法

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Oxidative protein folding is driven by the electron transport system

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CELL 1999年第2期98卷 217-227页

作者： Bader, M Muse, W Ballou, DP Gassner, C Bardwell, JCA Univ Michigan Dept Biol Ann Arbor MI 48109 USA Univ Michigan Dept Biol Chem Ann Arbor MI 48109 USA

Disulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron accepters, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron accepters such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system.

关键词：厌氧生活细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/代谢催化域细胞色素类/代谢 DNA引物二硫化物类/代谢电子转运/生理学电子传递链复合蛋白质类电子传递复合物Ⅳ/代谢大肠杆菌/化学大肠杆菌/酶学大肠杆菌/遗传学大肠杆菌蛋白质类膜蛋白质类/化学膜蛋白质类/遗传学膜蛋白质类/代谢诱变/生理学氧化还原氧化还原酶类/代谢氧/分析蛋白质二硫化物异构酶/化学蛋白质二硫化物异构酶/遗传学蛋白质二硫化物异构酶/代谢蛋白质折叠泛醌/代谢

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