BIOENERGETICS OF CYTOCHROME B-C1 COMPLEX

本研究项目的总体目标是研究质子 伴随细胞色素中电子转移的释放反应 bc 1，线粒体呼吸链区域。 最近我们 报道了二环己基碳二亚胺（DCCD）， 羧基修饰剂，阻断了质子的产电释放 在从酵母线粒体分离的BC复合物和BF复合物中 分离自菠菜叶绿体。 放射性标记的DCCD是 优先与bc 1复合物中的细胞色素B结合， BF复合物中的细胞色素b6，表明细胞色素B（b6）起作用， 在质子转移中的作用。 此外，DCCD共价结合至 天冬氨酸-160在酵母细胞色素B和天冬氨酸-152或 叶绿体细胞色素b6中的谷氨酸-165。 近期地形 基于亲水性图的B-细胞色素的预测具有 预测这些酸性残基位于两亲分子中， 螺旋（最初被认为是跨膜螺旋4）结合到 模型膜的外表面。 这意味着释放的质子 在醌醇底物的氧化过程中， 这种膜外螺旋到膜的细胞质侧， 这项拨款申请旨在解决螺旋4的作用， 细胞色素B在质子易位中的作用，具体目的如下：1） 为了确定细胞色素B在细胞内的位置， 线粒体膜和类囊体膜中的细胞色素b6 针对蛋白质区域的特异性抗体和选择性抗体 2）使用DCCD的荧光衍生物， 表征DCCD结合的羧基的环境， 3)研究突变体的酶活性和质子运动， 细胞色素B以建立参与这些过程的氨基酸， 和4）为了确定14 kDa蛋白，亚基7，的作用， bc 1复合物在电子转移和质子转移中的作用。

The overall goal of this research project is to investigate the proton releasing reactions that accompany electron transfer in the cytochrome bc1, region of the mitochondrial respiratory chain. Recently, we reported that dicyclohexylcarbodiimide (DCCD), the well-characterized carboxyl modifying reagent, blocked the electrogenic release of protons in the bc, complex isolated from yeast mitochondria and in a bf complex isolated from spinach chloroplasts. Radiolabeled DCCD was preferentially bound to cytochrome b in the bc1 complex and to cytochrome b6 in the bf complex suggesting that cytochrome b (b6) plays a role in proton translocation. Moreover, DCCD is covalently bound to aspartate-160 in yeast cytochrome b and to either aspartate-152 or glutamate-165 in the chloroplast cytochrome b6. Recent topographical projections for the b-cytochromes based on hydropathy plots have predicted that these acidic residues are localized in an amphipathic helix (initially proposed as membrane-spanning helix 4) bound to the outer surface of the model membrane. This implies that protons released during the oxidation of the quinol substrate are translocated through this extra-membranous helix to the cytoplasmic side of the membrane, This grant application proposes to address the role of helix 4 of cytochrome b in proton-translocation by the following specific aims: 1) To determine the topographical orientation of cytochrome b in the inner mitochondrial membrane and cytochrome b6 in the thylakoid membrane using specific antibodies against regions of the protein and selective proteolytic digestion, 2) To use fluorescent derivatives of DCCD to characterize the environment of the carboxyl group to which DCCD binds, 3) To study enzymatic activity and proton movements in mutants of cytochrome b to establish the amino acids involved in these processes, and 4) To establish the role of the 14 kDa protein, subunit 7, of the bc1 complex in electron transfer and proton translocation.