Substituent Of Respiratory Pathway And Mechanism Of Cytochrome C Maturation In Shewanella Oneidensis

Cytochromes c are widespread hemoproteins with covalently attached heme cofactors. This covalent attachment is processed by cytochrome c maturation (Ccm) system. Primarily, cytochromes c, as electron carriers or enzymes, are involved in energy transduction processes, such as respiration and photosynthesis. Shewanella oneidensis is a representative of a group of Gram-negative bacteria renowned for their respiratory versatility, which is largely attributed to abundant cytochromes c. This dissertation further studied the molecular mechanism of S. oneidensis nitrate and nitrite respiration based on the preliminary works. Two parts of important achivements had been obtained:1. Identified the substituent of quinol oxidase which delivered electrons to nitrate and nitrite reductases; 2. Elucidated the unique mechanism of the maturatioin of nitrite reductase NrfA.CymA, an inner-membrane tetraheme cytochrome c, is a quinol oxidase shared by multiple electron transport pathways in S. oneidensis. Loss of CymA compromises, but not abolishes, respiratory capabilities of many electron acceptors (EAs). It suggests there exists substituent(s) of CymA in anaerobic respiration pathways. A transposon random mutagenesis method was adopted to identify the substituent(s) of CymA in this study. A total of 87 inserted mutants in the background of AcymA were obtained, whose nitrate and nitrite respiration abilities were further reduced. After analyzing all of the inserted sites, petABC operon, which encodes cytochrome bc1 complex, was likely to be the candidate. Further study demonstrated both ubiquinone (UQ)and menaquinone (MK) served as electron pool for CymA and cytochrome bc1, despite of some preference. The further stronger ability of nitrate and nitrite respiration when cytochrome bc1 was absent should due to the increase of electron flow towards CymA, but not the increase of cymA transcription or the change of the ratio of UQ/MK. Furthermore, cytochrome bc1 transcription was affected by oxygen rather than nitrate or nitrite. At the same time, global regulators Crp and ArcA regulated cytochrome bc1 expression in an indirect way.Heme binding motifs (HBMs) of cytochromes c have been divided into two types: canonical and noncanonical. It has been reported that cytochrome c maturation (Ccm) system processes the maturation of canonical HBMs, while the maturation of noncanonical HBMs needs a dedicated maturation system. S. oneidensis nitrite reductase NrfA is a cytochrome c containing 4 canonical HBMs and 1 noncanonical HBM. This dissertation revealed the maturation of noncanonical HBM of NrfA absolutely depended on Ccm system but not a dedicated maturation system in S. oneidensis, even though the maturation of canonical HBM was still exclusively accomplished by Ccm system. In vivo and in vitro experiments indicated CcmⅠ was responsible for the unique cytochrome c maturation pattern of S. oneidensis. CcmⅠ was composed of insoluble transmembrane segment (CcmⅠ-1) and soluble periplasmic segment (CcmI-2). CcmⅠ was identified to be important but not essential for the maturation of canonical HBMs. On the contrary, the maturation of noncanonical HBM of NrfA was absolutely dependent on CcmⅠ. CcmI-1 was sufficient to fully restore the canonical HBMs maturation defect of Accml, however, CcmI-2 was dispensable. Neither parts of CcmⅠ was capable to fulfill the maturation of noncanonical HBM of NrfA, unless the intact CcmⅠ appeared. In vitro experiment demonstrated CcmI-2 interacted directly with NrfA, so we suggested CcmI-2 functioned as a chaperone for NrfA maturation. On the contrary, the role of CcmI-1 in NrfA maturation was unspecific. Ccml-1 ensured heme timely and proper attachment occured before the degradation of apoprotein.In conclusion, on one hand, we identified a substitute of CymA in respiratory pathway-cytochrome bc1. The molecular mechanism of electron transport pathway and regulation of cytochrome bc1 were studied intensively. The identification of cytochrome bc1 pathway filled the blank of respiratory network of S. oneidensis. On the other hand, Ccm component Ccml was identified to play distinct roles in canonical and noncanonical HBMs maturation for the first time. We elucidated the molecular mechanism of the maturation of nitrite reductase NrfA, which contains canonical and noncanonical HMBs.