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 oxoferryl-porphyrin 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(.-)(+) --> Trp(.-) (or Trp(.-)(+)) --> Tyr(.-). The M275I variant did not form the Trp(.-) 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.
Highlights
Catalase-peroxidases (KatGs)1 are present in bacteria and fungi and function primarily as hydrogen peroxide scavengers (1–3)
A mass spectrometric analysis of the tryptic peptides from recombinant wild-type KatG and the tryptophan, tyrosine, and methionine variants of Synechocystis (7) and B. pseudomallei (8) confirmed that this novel covalent adduct really exists in solution and may be a common feature to all catalase-peroxidases
The radical intermediates formed by the M275I, R439A, and R439N variants were investigated by using EPR spectroscopy and compared with those formed by the Y249F variant
Summary
KatG, catalase-peroxidase; CcP, cytochrome c peroxidase; APX, ascorbate peroxidase; CD, circular dichroism; EPR, electron paramagnetic resonance; HF EPR, high-field electron paramagnetic resonance. In comparison to wild-type KatG, these variants exhibit dramatic differences in both the catalase and the peroxidase activities, as well as the spectral features and kinetics of interconversion between their redox intermediates. Y249F variant shows spectral and kinetic features characteristic of most plant-type peroxidase intermediates, namely compounds I and II (11, 12), different from the wild-type KatG (10, 11). The Trp-Tyr-Met adduct appears to be hydrogen-bonded to a neighboring arginine residue (Arg439 in Synechocystis). In the H. marismortui and Synechococcus PCC 7942 KatG crystallographic structures, the corresponding arginine residue (Arg409) is in hydrogen-bonding distance to both Tyr218 and Met244 (Fig. 1A). In the crystal structure of B. pseudomallei KatG, the corresponding arginine (Arg426) points away from the covalent adduct in its predominant orientation (6), and no hydrogen bonds can be formed (Fig. 1B). The structural requirements that enable a peroxidase to disproportionate hydrogen peroxide are not really understood
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