Structural and Mutagenesis Studies on the Cytochrome c Peroxidase from Rhodobacter capsulatus Provide New Insights into Structure-Function Relationships of Bacterial Di-heme Peroxidases

Abstract

Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s ؊1 ), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Å in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies.

CcpA from Geobacter sulfurreducens Is a Basic Di-Heme Cytochrome c Peroxidase

“…17,19 In accordance with this finding, the analogous W97F variant of R. capsulatus CcpA showed less than 1% activity of the wild type, while a W97A variant was completely inactive. 21 Also, a W94A variant of the P. aeruginosa enzyme showed no activity in turnover experiments. 31 Another mutant of the P. aeruginosa enzyme, devoid of the entire loop 1 region 67-79, was also analyzed with respect to its activity.…”

“…Crystal structures are available for P. aeruginosa, 17 N. europaea, 18 Pseudomonas nautica, 19 P. pantotrophus 20 and R. capsulatus. 21 In contrast to eukaryotic peroxidases, CcpA proteins show a conserved tertiary structure consisting of two predominantly α-helical domains, each with one heme group covalently bound via thioether bonds to the cysteines of a binding motif, Cys-X 1 -X 2 -Cys-His. Due to the presence of two redox centers, the reduction of H 2 O 2 does not require the stabilization of a radical intermediate or the formation of an oxoferryl π-cation radical species (compound I), as each heme group is able to provide one electron to the substrate.…”

“…Between both hemes, a conserved Ca 2+ site has been linked to protein activity, and a highly conserved tryptophan residue was shown to be essential for H 2 O 2 reduction, as it is assumed to act as a key component of the electron transport pathway between both redox centers. 19,21 Depletion of this Ca 2+ has a strong effect on activity and it is assumed that the functional form of CcpA has this site fully occupied. 24 Electrons for the reduction of H 2 O 2 are derived from small, monoheme cytochromes c or alternatively from cupredoxins.…”

Mutation of Trp93 of MauG to tyrosine causes loss of bound Ca2+ and alters the kinetic mechanism of tryptophan tryptophylquinone cofactor biosynthesis

“…In fact, an overlay of the structures of MauG with those known for these DCCPs indicates that the positions and orientations of the two haems, the Ca 2 + , and the tryptophan residue are essentially identical [8]. This tryptophan residue has been proposed to act as an ET mediator between the two haems of the structurally characterized DCCPs from Pseudomonas aeruginosa, Nitrosomonas europaea, Pseudomonas nautica and Rhodobacter capsulatus [35][36][37][38]. However, its functional role has not been extensively studied.…”

Enzymatic activity mastered by altering metal coordination spheres

“…When calcium is not present, the side chain of this tryptophan residue is not in the proposed electron pathway between the two hemes. The importance of this residue in both the activation and catalytic activity of the enzyme has been shown through mutagenesis studies [47].…”