Structure-Function of Hetero-oligomeric Integral Membrane Proteins

描述(申请人提供)：细胞色素b6f复合体是少数(目前，17种)异寡体整膜蛋白中的一种，其晶体结构已被解析到3.0A以上。其结构是一种二聚体，它包裹着一个大的(10,000 A3)亲油性单体间的醌交换空腔，它与脂双层交换醌/对苯酚，并将一个单体电化学负N侧的醌还原中心与正P-侧[2Fe-2S]簇的氧化中心连接起来。N-端和p-端之间的对苯二酚途径涉及通过Ax！2a门脉。我们从嗜热蓝藻M.laminosus中解析了四个b6f结构，(I)一个天然结构，最近在Cd~(2+)存在下分辨率提高到2.95A，以及三个与苯醌类似物的配合物结构，p-侧TDS和DBMIB，以及n-侧NQNO。这些结构为苯二酚通过复合体并在其中扩散的路线提供了标记。进一步了解苯醌转移的结构和动力学对于理解疏水性药物和代谢物跨膜蛋白转移是相关的，并将通过更高分辨率的结构、定点突变和分子/导向动力学来追求。该复合体包含8个亚基：其中4个形成了结合8个假体基团的“大”亚基的核心。每个b6f单体包含4个亚铁血红素、一个[2Fe-2S]簇、1个质醌和3个独特的修饰基：(I)一个新的亚铁血红素，与细胞色素b的半胱氨酸残基有一个共价键，没有氨基酸侧链作为轴向配体；(Ii)一个叶绿素a，其中两个H2O已被分解为其第五个配体；(Iii)一个β-胡萝卜素，尽管它们14A分离，但CHI可以转移激发的三重态能量。这些b6f特有的假体基团对Be络合物的氧化还原功能提出了新的问题。我们将重点介绍独特的血红素，它的功能与b6f复合体进行铁还蛋白依赖的循环电子传递和光合膜释放O2的能力有关。在血红素和附近的(4A)血红素bn之间建立了一种独特的偶联，这意味着hemes bn-en具有作为2电子供体的能力，从而提供了一种新的机制来防止超氧化物和活性氧物种(ROS)的形成。蓝藻细胞膜的第二种异寡聚整膜蛋白NDH-1的研究也还原了苯二酚并产生ROS，其质量为14-15个亚基中的12个，其目标是获得晶体结构。

DESCRIPTION (provided by applicant): The cytochrome b6f complex is one of a small number (presently, 17) of hetero-oligomeric integral membrane proteins whose crystal structure has been solved to a resolution better than 3.0 A. The structure is a dimer that encloses a large (10,000 A3) lipophilic inter-monomer 'quinone exchange cavity that exchanges quinone/quinol with the lipid bilayer, and connects the quinone reduction site on the electrochemically negative n-side of one monomer with the oxidation site at the [2Fe-2S] cluster on the positive p-side. The quinone pathway between n- and p-sides involves passage through anllAx!2A portal. We have solved four b6f structures from the thermophilic cyanobacterium, M. laminosus, (i) a native structure with resolution recently improved to 2.95 A in the presence of Cd2+ cations, and three structures of complexes with quinone- analogue inhibitors, p-side TDS and DBMIB, and n-side NQNO. These structures have provided markers for the route of quinone passage across the complex and diffusion within it. Further understanding of the structure and dynamics of quinone transfer is relevant to understand transfer of hydrophobic drugs and metabolites across membrane proteins, and will be pursued through higher resolution structures, site-directed mutagenesis and molecular/steered dynamics. The complex contains 8 subunits: 4 of these form a core of 'large' subunits that bind 8 prosthetic groups. Each b6f monomer contains 4 hemes, one [2Fe-2S] cluster, one plastoquinone, and 3 unique prosthetic groups: (i) a novel heme en with one covalent linkage to a Cys residue of cytochrome b, and no amino acid side chains as axial ligands; (ii) one chlorophyll a, for which two H2O have been resolved as its 5th ligand; (iii) one beta-carotene to which the Chi, can transfer excited triplet state energy in spite of their 14A separation. These prosthetic groups unique to b6f raise new questions about redox function in be complexes. We will focus on the unique heme en, whose functions are linked to the ability of the b6f complex to carry out ferredoxin-dependent cyclic electron transport and of the photosynthetic membrane to evolve O2. A unique coupling between heme en and the nearby (4 A) heme bn was established, which implies an ability of hemes bn - en to serve as a 2 electron donor, thus providing a new mechanism to protect against formation of superoxide and reactive oxygen species (ROS). Studies on a second hetero- oligomeric integral membrane protein, Ndh-1 from the cyanobacterial membrane that also reduces quinone and generates ROS, have masses of 12 of the 14-15 subunits, and in which the goal is to obtain a crystal structure.