Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants.
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.
Department of Chemistry, Division of Biochemistry, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Christa
C
Jakopitsch
Anabella
A
Ivancich
Florian
F
Schmuckenschlager
Anuruddhika
A
Wanasinghe
Gerald
G
Pöltl
Paul Georg
PG
Furtmüller
Florian
F
Rüker
Christian
C
Obinger
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D003486:Cyanides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D007700:Kinetics; D013058:Mass Spectrometry; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology*
D001426:Bacterial Proteins; D003486:Cyanides; D005609:Free Radicals; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria