Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

Abstract

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp 122 , Tyr 249 , and Met 275 (Synechocysis KatG numbering). The known crystal structures suggest that Tyr 249 and Met 275 could be within hydrogen-bonding distance to Arg 439 . To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg 439 variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr 249 and Trp 122 . In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferrylporphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por ⅐ ؉ 3 Trp ⅐ (or Trp ⅐ ؉ ) 3 Tyr ⅐ . The M275I variant did not form the Trp ⅐ species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp 122 -Tyr 249 adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.Catalase-peroxidases (KatGs) 1 are present in bacteria and fungi and function primarily as hydrogen peroxide scavengers (1-3). Sequence similarities indicate they are members of the class I of the superfamily of plant, fungal, and bacterial peroxidases (4). In contrast to the other members of this group, such as cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), they show a high catalase activity comparable with monofunctional catalases. Although the catalase activity is overwhelming, KatGs also show a substantial peroxidase activity with various one electron donors.There are three crystal structures of KatGs available, namely the Archaebacterium Haloarcula marismortui (5), Burkholderia pseudomallei (6), and Synechococcus PCC 7942 (PDB entry 1UB2), the latter being a cyanobacterial KatG with high homology to Synechocystis KatG. The structures show that the arrangement of the active site is similar to CcP and APX, containing the distal triad arg...

Hydrogen peroxide oxidation by catalase‐peroxidase follows a non‐scrambling mechanism

“…This M-Y-W crosslink appears to be a characteristic common to all KatGs and has been demonstrated to be essential for the catalase activity [9,15,16,19]. Interestingly, this adduct can be associated with a KatG-typical arginine (R439) [2].…”

“…Interestingly, this adduct can be associated with a KatG-typical arginine (R439) [2]. Its association with the tyrosinate ion on the adduct is also required for optimum catalatic rates [19,23]. Another important residue at the distal heme cavity is D152, which is part of the substrate channel and has its side chain carboxyl group pointing toward the heme pocket.…”

Kinetics of Interconversion of Ferrous Enzymes, Compound II and Compound III, of Wild-type Synechocystis Catalase-peroxidase and Y249F

“…One significant structural difference between wild-type KatG and Y249F is that in the variant, the KatG-specific covalent adduct (which includes Tyr-249, Met-275, and Trp-122) at the distal heme side is absent (18). We now know three striking consequences; the catalase activity in Y249F is lost (whereas the peroxidase activity is unaffected), and in the presence of H 2 O 2 , an oxoferryl-type compound II is formed and easily converts to compound III (13,24). By contrast, in the presence of the covalent link, the catalase activity is high, and neither an oxoferryl-type compound I nor II accumulates, nor does compound III.…”

“…(i) Wild-type compound I produced with peroxoacetic acid reacts extremely slow with H 2 O 2 as demonstrated by the sequential stopped-flow technique (8,9). (ii) In wild-type Synechocystis catalase-peroxidase, the chemical nature of the intermediate referred to as conventional compound I was shown to be the superposition of the oxoferryl porphyrin -cation radical, the tryptophanyl radical, and the tyrosyl radical as demonstrated by our recent multifrequency EPR spectroscopy study (24). (iii) The observation of redox intermediates with unique features in their room temperature electronic absorption spectrum suggests the existence of alternative electronic structures of compound I (13,24).…”

“…Recent EPR experiments have shown the contribution of three different oxoferryl species obtained by reaction of wild-type KatG with peroxoacetic acid, namely an exchange-coupled porphyrin radical, a tryptophanyl, and a tyrosyl radical (13,24). Upon using H 2 O 2 in enzyme oxidation, neither the accumulation of the classical compound I (porphyrin radical-type, which typically shows a hypochromicity in the Soret peak) nor the accumulation of a protein radical species (which should exhibit an oxoferryl-type compound II spectrum like in cytochrome c peroxidase (27)) could be ever observed spectroscopically (in contrast to Y249F).…”