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The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE
Author(s) -
San Francisco Brian,
Sutherland Molly C.,
Kranz Robert G.
Publication year - 2014
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12510
Subject(s) - biochemistry , biology , cytochrome c , cytochrome , periplasmic space , cysteine , signal peptide , covalent bond , cytochrome c oxidase , peptide sequence , mitochondrion , enzyme , gene , chemistry , escherichia coli , organic chemistry
Summary Cytochrome c maturation (ccm) in many bacteria, archaea and plant mitochondria requires eight membrane proteins, CcmABCDEFGH , called system I . This pathway delivers and attaches haem covalently to two cysteines (of Cys ‐ X xx‐ X xx‐ Cys ‐ His ) in the cytochrome c . All models propose that CcmFH facilitates covalent attachment of haem to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH . We report formation of holocytochromes c by CcmFH and CcmG , a periplasmic thioredoxin, independent of CcmABCDE (we term this activity CcmFGH ‐only). Cytochrome c produced in the absence of CcmABCDE is indistinguishable from cytochrome c produced by the full system I , with a cleaved signal sequence and two covalent bonds to haem. We engineered increased cytochrome c production by CcmFGH ‐only, with yields approaching those from the full system I . Three conserved histidines in CcmF ( TM ‐ His 1, TM ‐ His 2 and P ‐ His 1) are required for activity, as are the conserved cysteine pairs in CcmG and CcmH . Our findings establish that CcmFH is the system I holocytochrome c synthetase. Although we discuss why this engineering would likely not replace the need for CcmABCDE in nature, these results provide unique mechanistic and evolutionary insights into cytochrome c biosynthesis.