Structure-function analysis of the heme-binding WWD domain in the bacterial holocytochrome c synthase, CcmFH.

Heme trafficking is a fundamental biological process, yet its direct study has been hampered due to heme's tight intracellular regulation, heme cytotoxicity, and the transient nature of trafficking. The bacterial System I and System II cytochrome c biogenesis pathways are developing into models to interrogate heme trafficking mechanisms, as they function to transport heme from inside to outside the cell for attachment to apocytochrome c . Cytochromes c require heme for folding and to function in the context of electron transport chains for critical cellular functions, such as respiration. We focus on System I, comprised of eight membrane proteins, CcmABCDEFGH, proposed to function in two steps: CcmABCD mediates the transfer of heme and attachment to CcmE. HoloCcmE chaperones heme to CcmFH for attachment to apocytochrome c . While CcmFH is known to be the holocytochrome c synthase, the mechanism of heme interaction and positioning for attachment to apocytochrome c remains to be elucidated. A comprehensive structure-function analysis of the conserved WWD domain in CcmF was undertaken utilizing alanine-scanning and cysteine-scanning, revealing residues critical for CcmF's synthase function and residues required for interaction with the 2- and 4-vinyls of heme. This analysis demonstrates for the first time that the CcmF WWD domain directly interacts with heme and that heme interactions within this domain are required for attachment to apocytochrome c . This in-depth interrogation of heme binding now allows for comparison across cytochrome c biogenesis proteins CcmF, CcmC, and CcsBA, revealing common mechanisms of heme interaction in these heme trafficking pathways.IMPORTANCEHeme is an essential co-factor for proteins involved with critical cellular functions, such as energy production and oxygen transport. Thus, understanding how heme interacts with proteins and is moved through cells is a fundamental biological question. This work studies the System I cytochrome c biogenesis pathway, which in some species (including Escherichia coli ) is composed of eight integral membrane or membrane-associated proteins called CcmA-H that are proposed to function in two steps to transport and attach heme to apocytochrome c . Cytochrome c requires this heme attachment to function in electron transport chains to generate cellular energy. A conserved WWD heme-handling domain in CcmFH is analyzed and residues critical for heme interaction and holocytochrome c synthase activity are identified. CcmFH is the third member of the WWD domain-containing heme-handling protein family to undergo a comprehensive structure-function analysis, allowing for comparison of heme interaction across this protein family.