Architecture of the Heme-translocating CcmABCD/E complex required for Cytochrome c maturation.

Mono- and multiheme cytochromes c are post-translationally matured by the covalent attachment of heme. For this, Escherichia coli employs the most complex type of maturation machineries, the Ccm-system (for cytochrome c maturation). It consists of two membrane protein complexes, one of which shuttles heme across the membrane to a mobile chaperone that then delivers the cofactor to the second complex, an apoprotein:heme lyase, for covalent attachment. Here we report cryo-electron microscopic structures of the heme translocation complex CcmABCD from E. coli, alone and bound to the heme chaperone CcmE. CcmABCD forms a heterooctameric complex centered around the ABC transporter CcmAB that does not by itself transport heme. Our data suggest that the complex flops a heme group from the inner to the outer leaflet at its CcmBC interfaces, driven by ATP hydrolysis at CcmA. A conserved heme-handling motif (WxWD) at the periplasmic side of CcmC rotates the heme by 90° for covalent attachment to the heme chaperone CcmE that we find interacting exclusively with the CcmB subunit.

Architecture of the membrane-bound cytochrome c heme lyase CcmF.

The covalent attachment of one or multiple heme cofactors to cytochrome c protein chains enables cytochrome c proteins to be used in electron transfer and redox catalysis in extracytoplasmic environments. A dedicated heme maturation machinery, whose core component is a heme lyase, scans nascent peptides after Sec-dependent translocation for CXnCH-binding motifs. Here we report the three-dimensional (3D) structure of the heme lyase CcmF, a 643-amino acid integral membrane protein, from Thermus thermophilus. CcmF contains a heme b cofactor at the bottom of a large cavity that opens toward the extracellular side to receive heme groups from the heme chaperone CcmE for cytochrome maturation. A surface groove on CcmF may guide the extended apoprotein to heme attachment at or near a loop containing the functionally essential WXWD motif, which is situated above the putative cofactor binding pocket. The structure suggests heme delivery from within the membrane, redefining the role of the chaperone CcmE.

Structural and Functional Analysis of Latex Clearing Protein (Lcp) Provides Insight into the Enzymatic Cleavage of Rubber.

Latex clearing proteins (Lcps) are rubber oxygenases that catalyse the extracellular cleavage of poly (cis-1,4-isoprene) by Gram-positive rubber degrading bacteria. Lcp of Streptomyces sp. K30 (LcpK30) is a b-type cytochrome and acts as an endo-type dioxygenase producing C20 and higher oligo-isoprenoids that differ in the number of isoprene units but have the same terminal functions, CHO-CH2- and -CH2-COCH3. Our analysis of the LcpK30 structure revealed a 3/3 globin fold with additional domains at the N- and C-termini and similarities to globin-coupled sensor proteins. The haem group of LcpK30 is ligated to the polypeptide by a proximal histidine (His198) and by a lysine residue (Lys167) as the distal axial ligand. The comparison of LcpK30 structures in a closed and in an open state as well as spectroscopic and biochemical analysis of wild type and LcpK30 muteins provided insights into the action of the enzyme during catalysis.