Heme Redox Potential Control in de Novo Designed Four-R-Helix Bundle Proteins †
Heme Redox Potential Control in de Novo Designed Four-R-Helix Bundle Proteins †
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从头设计的四 R 螺旋束蛋白中的血红素氧化还原电位控制 †
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通讯作者：
P. Dutton
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作者：
Julia M. Shifman;B. Gibney;R. Sharp;P. Dutton
使用稳健、特征良好的血红素蛋白模型、合成蛋白支架 H10A24 [{CH3CONH-CGGGELWKL‚HEELLKK ‚FEELLKL‚AEERLKK‚L-CONH2}2]2 通过实验研究了金属卟啉还原电位调节的各种机制的影响。从高介电水环境中去除铁卟啉大环并隔离在 H10A24 模型的疏水核心内，将平衡还原中点电势提高了 36-138 mV，具体取决于金属卟啉结构的疏水性。通过将各种天然和合成的金属卟啉整合到单个蛋白质支架中，由于外围大环取代基的给电子/吸电子特性，我们证明了还原电位调节的范围为 300 mV。无论大环结构如何，通过控制参与铁血红素部分电荷补偿的谷氨酸的质子化状态，溶液 pH 值用于将金属卟啉还原电位调节 160 mV。通过将带电氨基酸插入靠近金属卟啉的疏水核心来控制还原电位的尝试取得了不同的成功，H10A24-L13E 将 Em8.5 降低了 40 mV，H10A24-E11Q 将其提高了 50 mV，而 H10A24-L13R 令人惊讶地保持不变。修改相邻金属卟啉的电荷，铁(III)原卟啉IX为+1或锌(II)原卟啉IX为中性，导致模型中观察到的70 mV [Fe(III)PPIX]+ [Fe(III)PPIX]+相互作用损失。结合使用这些因素，我们说明了简单血红素模型中金属卟啉还原中点电位的 435 mV 变化，相对于天然细胞色素观察到的约 800 mV 范围。血红素模型和其他从头设计的血红素蛋白的还原电位之间的比较揭示了合成细胞色素的 Em 值的总体趋势。氧化还原蛋白利用相对少量的辅因子来执行多种任务。因此，生物辅助因子通常表现出广泛的氧化还原活性，每个特定的辅助因子都调整到促进蛋白质功能的范围。辅因子还原电位的巨大变化是通过周围各向异性蛋白质基质对氧化还原中心施加的各种宏观和微观环境影响来实现的。血红素（Fe 原卟啉 IX 及其衍生物）形成一类公认的生物辅助因子，是细胞色素主导的蛋白质家族的功能中心。虽然细胞色素的氧化/还原总是涉及看似简单的从血红素铁转移电子到血红素铁的电子转移反应，但它们的还原中点电位跨越 800 mV 范围，从细胞色素 c3（-400 mV vs SHE）到细胞色素 b559（+400 mV）(2)（方案 1）。金属卟啉还原电位的调节受到铁轴向连接的性质、卟啉外围取代基、金属位点的溶剂可及性、与蛋白质侧链和其他辅助因子的静电相互作用以及邻近氨基酸的质子化状态的影响。虽然决定氧化还原活性的因素已在天然血红素蛋白中进行了实验和理论研究 (3-8)，但从头设计了血红素蛋白模型，即含有生化辅因子的合成蛋白支架 (9)，为研究血红素蛋白还原中点电位控制的基础提供了建设性方法。在目前的工作中，构建了一系列水溶性血红素蛋白模型，即具有以细胞色素bc1复合物（10-13）的细胞色素b亚基为模型的血红素结合位点的合成四R螺旋束，以纳入各种微观和宏观环境因素。使用这一系列模型，我们研究了血红素外周取代因素、与血红素附近带电氨基酸以及其他血红素辅因子的静电相互作用以及邻近氨基酸的质子化/去质子化等因素对合成蛋白质氧化还原活性调节的程度（图1）。因此，我们在原型合成血红素蛋白模型系统中实现了血红素还原中点电位 (435 mV) 的显着多样性。材料和方法 三氟乙酸、乙醚、乙酸酐、哌啶和吡啶获自 Aldrich † 这项工作得到了美国公共卫生服务拨款 GM48130 的支持。 * 通讯作者。电话：(215)898-5668。传真：(215)573-2235。电子邮件：dutton@mail.med.upenn.edu。 ‡ 目前地址：Division of Biology, California Institute of Technology, 147-75, Pasadena, CA 19125。 § 目前地址：Department of Chemistry, Columbia University, New York, NY 10027. 14813 Biochemistry2000,39, 14813-14821 10.1021/bi000927b CCC：$19.00 © 2000 年美国化学会于 2000 年 4 月 11 日在 Web 上发布 Chemical Co.（密尔沃基，威斯康星州）。乙二硫醇和 1-羟基苯并三唑 (HOBt) 1 购自 Fluka (Ronkonkoma, NY)。血红素购自卟啉产品公司（洛根，犹他州）。 NovaSyn PR-500 树脂购自 Calbiochem-Novabiochem (La Jolla, CA)。天然 Fmoc 保护的氨基酸以五氟苯酯形式从 PerSeptive Biosystems (Framingham, MA) 获得，但 FmocL-Arg(Pmc)-OPfp 除外，FmocL-Arg(Pmc)-OPfp 是从 Bachem (King of Prusus, PA) 购买的。使用从 Pierce (Rockford, IL) 收到的盐酸胍 (8 M)。氧化还原介体染料购自 Aldrich Chemical Co.（威斯康星州密尔沃基），但绿脓素除外，绿脓素是由从 Fluka（纽约州朗康科马）获得的 N-甲基吩氮鎓硫酸甲酯合成的。所有其他化学品和溶剂均为试剂级。方案 1：天然和合成细胞色素的氧化还原活性等级 a a 显示水溶液中游离氯化血红素 FePPIXCl 的还原中点电位以进行比较。还原中点电位值对应于 pH 7 -8 范围。氧化还原活性值的参考文献：cyt b559 (2)、细胞色素b6 f 复合物的细胞色素f (57)、细胞色素c氧化酶的细胞色素A和细胞色素A3 (28)、细胞色素c (58)、细胞色素b562(59)、细胞色素bc1复合物的细胞色素bL和bH (60)、细胞色素b5 (61)、辣根过氧化物酶( 62)、细胞色素 P-450 ( 63)、TASP 设计的蛋白质 di-FePPIX MOP1 ( 47)、设计的 FePPIX 蛋白质 Retro(S-S) 和 VAVH25 (46)、自组装肽 FeCPI-FF-Aib ( 51) 、肽夹心中间血红素 (FeMPIX-PSM) ( 52)。图 1：FePPIX 2-H10A24 血红素蛋白模型和本研究中使用的各种金属卟啉衍生物的分子模型表示。使用 SYBYL（Tripos Associates，圣路易斯，密苏里州）在 Silicon Graphics Indigo 2 工作站（加利福尼亚州山景城）上进行建模。 14814 生物化学，卷。 39，第 48 期，2000 年希夫曼等人。
The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [ {CH3CONH-CGGGELWKL‚HEELLKK ‚FEELLKL‚AEERLKK‚L-CONH2}2]2. Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the Em8.5 by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX]+ [Fe(III)PPIX]+ interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in theEm values of synthetic cytochromes. Redox proteins utilize a relatively small number of cofactors to perform a multitude of tasks. Consequently, it is common for a biological cofactor to display a wide range of redox activity, with each particular cofactor tuned into the range that facilitates protein function. Large variations in cofactor reduction potentials are achieved through a variety of macroand microenvironmental effects imposed on the redox centers by the surrounding anisotropic protein matrix. Hemes (Fe protoporphyrin IX and its derivatives) form a well-recognized class of biological cofactors that are the functional centers in a family of proteins dominated by the cytochromes. While oxidation/reduction of cytochromes invariably involves a seemingly simple reaction of electron transfer from/to the heme iron, their reduction midpoint potentials span an 800-mV range, from cytochrome c3 (-400mV vs SHE) to cytochromeb559 (+400 mV) (2) (Scheme 1). The modulation of metalloporphyrin reduction potentials is influenced by the nature of the axial ligation to the iron, porphyrin peripheral substituents, solvent accessibility of the metal site, electrostatic interactions with protein side chains and other cofactors, and protonation state of neighboring amino acids. While the factors determining redox activity have been both experimentally and theoretically studied in natural heme proteins ( 3-8), de novo designed heme protein maquettes , synthetic protein scaffolds containing biochemical cofactors (9), offer a constructive approach to the study of the foundations of heme protein reduction midpoint potential control. In the present work, a family of water-soluble heme protein maquettes, synthetic fourR-helix bundles with hemebinding sites modeled after the cytochrome b subunit of the cytochromebc1 complex (10-13) have been constructed to incorporate various microand macroenvironmental factors. Using this series of maquettes, we investigate the magnitude of redox activity regulation in synthetic proteins by the factors of heme peripheral substitution, electrostatic interactions with charged amino acids in heme vicinity as well as with other heme cofactors, and protonation/deprotonation of neighboring amino acids (Figure 1). As a result, we achieve significant diversity in the heme reduction midpoint potential (435 mV) within our prototype synthetic heme protein maquette system. MATERIALS AND METHODS Trifluoroacetic acid, diethyl ether, acetic anhydride, piperidine, and pyridine were obtained from the Aldrich † This work was supported by U.S. Public Health Service Grant GM48130. * Corrresonding author. Phone: (215)898-5668. Fax: (215)573-2235. E-mail: dutton@mail.med.upenn.edu. ‡ Present address: Division of Biology, California Institute of Technology, 147-75, Pasadena, CA 19125. § Present address: Department of Chemistry, Columbia University, New York, NY 10027. 14813 Biochemistry2000,39, 14813-14821 10.1021/bi000927b CCC: $19.00 © 2000 American Chemical Society Published on Web 11/04/2000 Chemical Co. (Milwaukee, WI). Ethanedithiol and 1-hydroxybenzotriazole (HOBt) 1 were purchased from Fluka (Ronkonkoma, NY). Hemin was purchased from Porphyrin Products Inc. (Logan, UT). The NovaSyn PR-500 resin was purchased from Calbiochem-Novabiochem (La Jolla, CA). Natural Fmoc-protected amino acids were acquired as pentafluorophenyl esters from PerSeptive Biosystems (Framingham, MA) with the exception of FmocL-Arg(Pmc)-OPfp, which was purchased from Bachem (King of Prussia, PA). Guanidine hydrochloride (8 M) was used as received from Pierce (Rockford, IL). Redox mediator dyes were purchased from Aldrich Chemical Co. (Milwaukee, WI) with the exception of pyocyanine, which was synthesized from N-methylphenazonium methosulfate obtained from Fluka (Ronkonkoma, NY). All other chemicals and solvents were reagent grade. Scheme 1: Redox Activity Scale of Natural and Synthetic Cytochromes a a The reduction midpoint potential of free hemin, FePPIXCl, in aqueous solution is shown for comparison. Reduction midpoint potential values correspond to pH 7 -8 range. References for redox activity values: cyt b559 (2), cytochromef of cytochromeb6 f complex (57), cytochromeA and cytochromeA3 of cytochromec oxidase ( 28), cytochromec (58), cytochromeb562(59), cytochromebL and bH of cytochromebc1 complex (60), cytochromeb5 (61), horseradish peroxidase ( 62), cytochrome P-450 ( 63), TASP designed proteins di-FePPIX MOP1 ( 47), designed FePPIX proteins retro(S-S) and VAVH25 (46), self-assembled peptide FeCPI-FF-Aib ( 51), peptide sandwiched mesoheme (FeMPIX-PSM) ( 52). FIGURE 1: Molecular modeling representation of the FePPIX 2-H10A24 heme protein maquette and the various metalloporphyrins derivatives utilized in this study. Modeling was performed on a Silicon Graphics Indigo 2 w rkstation (Mountain View, CA) using SYBYL (Tripos Associates, St. Louis, MO). 14814 Biochemistry, Vol. 39, No. 48, 2000 Shifman et al.