PROBING THE ACTIVE SITE OF THE MITOCHONDRIAL HOLOCYTOCHROME C SYNTHASE, HCCS

描述(申请人提供)：在真核生物中，细胞色素c在有氧呼吸和细胞凋亡中都起着至关重要的作用，从而影响细胞的生死。C型细胞色素的特征是共价结合一个血红素辅助因子，这是其稳定性和后续功能所必需的修饰。血红素附着发生在线粒体中，由全细胞色素c合成酶(HcCS)介导，全细胞色素c合成酶是真核细胞色素c生物发生途径的主要组成部分，也称为系统III。 先前在动物身上的研究表明，HCCS的缺陷可导致致命性或人类疾病小眼球伴线性皮肤缺陷(MLS)。尽管在酵母中发现HCCS已有25年的历史，但其功能机制尚不清楚，这主要是由于其缺乏重组表达和不稳定性所致。我们的实验室最近成功地克服了这些技术限制，使我们能够启动对HCCS结构/功能的第一次全面生化分析。 HCCS的催化功能依赖于其与底物、血红素和细胞色素c相互作用和协调的能力。因此，本研究试图确定HCCS中的哪些残基和/或结构域构成其活性部位。目的1通过检测血红素结合和细胞色素c募集和成熟的扰动，分析HCCS中高度保守残基突变的功能后果。在AIM 2中，通过使用含有血红素附着位点的可UV交联的细胞色素c肽，直接评估了HCCS的凋亡素c底物结合部位。 这些研究将提供第一次深入的机制分析，从而促进我们对线粒体生物能量学的理解。我们的结果将直接影响目前已知的由HCCS异常引起的疾病，如MLS，并有助于了解在其他人类条件下观察到的线粒体许多故障。

DESCRIPTION (provided by applicant): In eukaryotes, cytochrome c plays a vital role in both aerobic respiration and apoptosis, thus impacting the life and death of a cell. C-type cytochromes are characterized by covalent attachment of a heme co-factor, a modification that is required for its stability and subsequent function. Heme attachment takes place in the mitochondria and is mediated by holocytochrome c synthase, HCCS, the primary component of the eukaryotic cytochrome c biogenesis pathway, also known as System III. Previous studies in animals have shown that defects in HCCS can result in lethality or the human disease microphthalmia with linear skin defects (MLS). Although HCCS was discovered in yeast 25 years ago, the mechanisms underlying HCCS function have yet to be explored, largely due to its poor recombinant expression and instability. Our lab has very recently been successful in overcoming these technical limitations, allowing us to initiate the first comprehensive biochemical analysis of HCCS structure/ function. The catalytic function of HCCS depends on its ability to interact with and coordinate interactions between its substrates, heme and cytochrome c. Therefore, the proposed study seeks to determine which residues and/or domains in HCCS comprise its active site. Aim 1 analyzes the functional consequences of mutations of highly conserved residues in HCCS by examining perturbations in heme binding and cytochrome c recruitment and maturation. The apocytochrome c substrate-binding site of HCCS is directly assessed in Aim 2 by peptide crosslinking, using a UV-crosslinkable cytochrome c peptide containing the heme-attachment site. These studies will provide the first in-depth mechanistic analysis of HCCS, thus advancing our understanding of mitochondrial bioenergetics. Our results will directly impact what is currently known about diseases like MLS that are caused by HCCS abnormalities, as well as contribute to the body of knowledge concerning the many malfunctions observed in mitochondria in other human conditions.