Protein-Based Radicals in the Catalase-Peroxidase of Synechocystis PCC6803: A Multifrequency EPR Investigation of Wild-Type and Variants on the Environment of the Heme Active Site

Citations Over TimeTop 1% of 2003 papers

Abstract

Catalase-peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 catalase-peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to the heme and spanning the distal and proximal sides were also investigated. In the wild-type enzyme, the EPR signal of the catalases and peroxidases (typical) Compound I intermediate [Fe(IV)=O por.+] was observed. Two protein-based radical intermediates were also detected and identified as a Tyr. and a Trp. . The site of Trp. is proposed to be Trp 106, a residue belonging to the conserved short stretch in catalase-peroxidases and located at a 7-8 A distance to the heme propionate groups. An extensive hydrogen-bonding network on the heme distal side, involving Trp122, His123, Arg119, seven structural waters, the heme 6-propionate group, and Trp106, is proposed to have a key role on the formation of the tryptophanyl radical. We used high-field EPR spectroscopy (95-285 GHz) to resolve the g-anisotropy of the protein-based radicals in Synechocystis catalase-peroxidase. The broad gx component of the HF EPR spectrum of the Tyr. in Synechocystis catalase-peroxidase was consistent with a distributed electropositive protein environment to the tyrosyl radical.

Related Papers

• → Converting Cytochrome c into a Peroxidase‐Like Metalloenzyme by Molecular Design(2007)34 cited
• → Reconstitution of Heme Enzymes with Artificial Metalloporphyrinoids(2016)16 cited
• → Structural Measurements in Hemoproteins: Use of Spin-Labeled Protoheme as a Probe of Heme Environment(1971)24 cited
• → Incorporation of Modified and Artificial Cofactors into Naturally Occurring Protein Scaffolds(2014)2 cited
• Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp. PCC 6803(2008)