Engineering the proximal heme cavity of catalase-peroxidase.
Catalase-peroxidases (KatGs) are prokaryotic heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs, CCP and APXs contain identical amino acid triads in the heme pocket (distal Arg/Trp/His and proximal His/Trp/Asp), but differ dramatically in their reactivities towards hydrogen peroxide and various one-electron donors. Only KatGs have high catalase activity in addition to a peroxidase activity of broad specificity. Here, we investigated the effect of mutating the conserved proximal triad on KatG catalysis. With the exception of W341F, all variants (H290Q, W341A, D402N, D402E) exhibited a catalase activity <1% of wild-type KatG and spectral properties indicating alterations in heme coordination and spin states. Generally, the peroxidase activity was much less effected by these mutations. Compared with wild-type KatG the W341F variant had a catalase and halogenation activity of about 40% and an even increased overall peroxidase activity. This variant, for the first time, allowed to monitor the hydrogen peroxide mediated transitions of ferric KatG to compound I and back to the resting enzyme. Compound I reduction by aromatic one-electron donors (o-dianisidine, pyrogallol, aniline) was not influenced by exchanging Trp by Phe. The findings are discussed in comparison with the data known from CCP and APX and a reaction mechanism for the multifunctional activity of the W341F variant is suggested.
Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Christa
C
Jakopitsch
Günther
G
Regelsberger
Paul Georg
PG
Furtmüller
Florian
F
Rüker
Günter A
GA
Peschek
Christian
C
Obinger
D000975:Antioxidants / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism; D001426:Bacterial Proteins; D000458:Cyanobacteria / Q000201:enzymology; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D010463:Peracetic Acid / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000975:Antioxidants; D001426:Bacterial Proteins; D006420:Hemeproteins; D016877:Oxidants; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; D010463:Peracetic Acid; D001205:Ascorbic Acid