Characterization of cytochrome C synthetase CcsBA and latex clearing protein

Abstract: The c-type cytochromes are a wide group of metalloproteins, characterized by the covalent attachment of the heme group to the surrounding polypeptide. Their assembly requires assistance from dedicated systems to bring together and to catalyse the thioether bond formation between the vinyl groups of the heme group and two cysteine residues found in a signature motif, most common CXXCH. At least three maturation pathways were developed during evolution [1]. System II or cytochrome c synthesis (Ccs), found in several bacterial species and in chloroplasts, comprises two proteins responsible for the reduction of the heme binding motif on the apocytochrome c and CcsBA complex, dedicated for cytochrome c production. CcsA displays a high conserved region, referred as the WWD motif, and together with two flanking histidine residues orients and positions the heme. The first part of the current study was focused on screening different homologs of CcsBA (or just CcsA) by heterologous production, purification and crystallization in order to elucidate the three-dimensional structure via X-ray crystallography. The CcsBA/CcsA homologs were isolated with a b-type heme found in an oxidized state (Fe3+), indicated by UV/Vis spectral analysis. CcsBA fusion protein from Bacteroides thetaiotaomicron in complex with n-Dodecyl β-d-maltoside (DDM) was the only homolog successfully crystallized. However, the crystal optimization by intensive screening of different conditions did not improve the quality of the diffraction experiments.
The second part of this thesis focuses on the structural characterization by X-ray crystallography of latex clearing protein (Lcp) from Streptomyces sp. strain K30, a b-type cytochrome involved in rubber biodegradation. The crystallized structure of Lcp was solved by exploiting the anomalous signal of the heme iron. The three-dimensional model revealed a globular protein, with the core of the protein structurally related to the members of the globin family. Two different structural conformations were observed depending on the nature of the sixth axial ligand of the iron heme. In the close state, a lysine residue from the protein’s backbone unusually binds the iron atom in the distal site, suggesting a role in the enzymatic reaction, while in the open state the same position is occupied by an imidazole molecule, emerged from the crystal growth condition. Additional electron paramagnetic resonance experiments on LcpK30 were conducted to investigate intermediate states of the heme group

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2018

Classification and Topics

• Keyword: Cytochrome c

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2018
• Creator: Ilcu, Lorena
• Contributor: Einsle, Oliver ; Albert-Ludwigs-Universität Freiburg. Fakultät für Chemie und Pharmazie

Further information

• DOI: 10.6094/UNIFR/17125
• URN: urn:nbn:de:bsz:25-freidok-171257
• Rights: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 25.03.2025, 3:52 AM CET