Characterisation of haem lyases and their dedicated roles in bacterial cytochrome c biogenesis

背景：c型细胞色素是一类广泛存在的蛋白质，在原核生物和真核生物（包括许多病原体）的能量保存代谢中起着至关重要的作用。细胞色素c生物发生的关键特征是血红素辅助因子与血红素c结合基序中两个（或很少只有一个）半胱氨酸残基的共价附着，通常是CXXCH。这个反应是由膜结合酶血红素裂解酶催化的。目前还没有纯化过细菌血红素裂解酶，其反应机理也不清楚。催化细菌细胞色素c生物生成的两个独立的酶系统已经被描述过，但只有大肠杆菌的Ccm系统（或系统I）被详细研究过。目的：本项目旨在揭示参与CcsA系统（或系统II）的血红素裂解酶的功能。可以说最适合的模式细菌是琥珀酸Wolinella succinogenes。它的基因组编码三种不同的血红素裂解酶，这些酶对不同的血红素c结合基序的特异性不同。我们还将研究琥珀酸芽孢杆菌野生型和血红素裂解酶基因缺失突变体是否能够将血红素共价附着在含有经典或各种非常规血红素c结合基元的模型c型细胞色素上。一种血红素裂解酶（NrfI）已经被证明可以识别五血红素细胞色素c亚硝酸盐还原酶的独特CXXCK血红素c结合基元。为了鉴定必需氨基酸残基，NrfI将进行位点定向修饰。系统II血红素裂解酶的过量生产、纯化和生化特性将进行尝试。影响：这项工作将扩大对翻译后蛋白成熟和血红素c结合基序预测的一般认识。此外，还旨在建立琥珀酸芽孢杆菌作为高效生产c型细胞色素的宿主。关键词：细胞色素c成熟；细胞色素c血红素裂解酶；血红素c结合基序；Wolinella succinogenes;细菌能量代谢。

Background: C-type cytochromes are a widespread class of proteins that play a vital role in the energy-conserving metabolism of prokaryotic and eukaryotic organisms including many pathogens. The key feature in cytochrome c biogenesis is the covalent attachment of the haem cofactor to two (or rarely only one) cysteine residues arranged in a haem c binding motif, typically CXXCH. This reaction is catalysed by the membranebound enzyme haem lyase. No bacterial haem lyase has ever been purified and its reaction mechanism is unknown. Two independent enzymic systems catalysing bacterial cytochrome c biogenesis have been described but only the Ccm system (or system I) of Escherichia coli has been investigated in any detail. Goals: This project aims to reveal the function of haem lyases involved in the CcsA system (or system II). The arguably best-suited model bacterium is Wolinella succinogenes. Its genome encodes three distinct haem lyases that are expected to differ in their specificity towards distinct haem c binding motifs. It will be examined whether or not W. succinogenes wild-type and haem lyase gene deletion mutants are able to covalently attach haem to a model c-type cytochrome containing either the classical or various unconventional haem c binding motifs. One haem lyase (NrfI) has already been shown to recognise a unique CXXCK haem c binding motif of pentahaem cytochrome c nitrite reductase. NrfI will be subject to site-directed modification in order to identify essential amino acid residues. Overproduction, purification and biochemical characterisation of system II haem lyases will be attempted. Impact: The proposed work will broaden the general knowledge on posttranslational protein maturation and prediction of haem c binding motifs. Furthermore, it aims to establish W. succinogenes as host for efficient c-type cytochrome production. Key words: Cytochrome c maturation; Cytochrome c haem lyase (CCHL); Haem c binding motif; Wolinella succinogenes; Bacterial energy metabolism.