The catalytic role of the distal site asparagine‐histidine couple in catalase‐peroxidases

Abstract

Catalase-peroxidases (KatGs) are unique in exhibiting an overwhelming catalase activity and a peroxidase activity of broad specificity. Similar to other peroxidases the distal histidine in KatGs forms a hydrogen bond with an adjacent conserved asparagine. To investigate the catalytic role(s) of this potential hydrogen bond in the bifunctional activity of KatGs, Asn153 in Synechocystis KatG was replaced with either Ala (Asn153fiAla) or Asp (Asn153fiAsp). Both variants exhibit an overall peroxidase activity similar with wild-type KatG. Cyanide binding is monophasic, however, the second-order binding rates are reduced to 5.4% (Asn153fiAla) and 9.5% (Asn153fiAsp) of the value of native KatG [(4.8 ± 0.4) · 10 5 M )1 AEs )1 at pH 7 and 15°C]. The turnover number of catalase activity of Asn153fiAla is 6% and that of Asn153fiAsp is 16.5% of wild-type activity. Stopped-flow analysis of the reaction of the ferric forms with H 2 O 2 suggest that exchange of Asn did not shift significantly the ratio of rates of H 2 O 2 -mediated compound I formation and reduction. Both rates seem to be reduced most probably because (a) the lower basicity of His123 hampers its function as acid-base catalyst and (b) Asn153 is part of an extended KatG-typical H-bond network, the integrity of which seems to be essential to provide optimal conditions for binding and oxidation of the second H 2 O 2 molecule necessary in the catalase reaction.Keywords: catalase-peroxidase; Synechocystis PCC 6803; catalase activity; peroxidase activity; compound I.On the basis of sequence similarities with yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs), catalase-peroxidases (KatGs) have been shown to be members of class I of the superfamily of plant, fungal and bacterial heme peroxidases [1]. KatGs have been found in prokaryotes (archaebacteria and eubacteria) and fungi and are homomultimeric proteins with monomers being twice as large as CCP or APXs adding up to about 79-85 kDa, which is ascribed to gene duplication [2]. From both CCP and APX the crystal structures have been solved [3,4] and, quite recently, the 2.0 Å crystal structure of the homodimeric KatG from Haloarcula marismortui has been published [5]. This structure and sequence alignments suggest that all class I peroxidases have conserved the amino-acid triad His, Asp and Trp in the proximal pocket and the triad Trp, Arg and His in the distal pocket (Fig. 1). Despite this homology, class I peroxidases dramatically differ in their reactivities towards hydrogen peroxide and one-electron donors. Catalase-peroxidases have a predominant catalase activity but differ from monofunctional catalases in also exhibiting a substantial peroxidatic activity with broad specificity. However, no substantial catalase activity has ever been reported for either CCP or APX. Cytochrome c peroxidase (CCP) is unusual in that it prefers another protein (cytochrome c) as a redox partner, whereas ascorbate peroxidases (APXs) prefer the anion ascorbate as electron donor. But both cytochrome c and ascorba...

Diversity of structures and properties among catalases

“…The ready accumulation of a compound I radical was demonstrated, confirming the importance of the distal Trp in the second stage of the catalatic reaction and its lack of importance in the first-stage formation of compound I. More recently, the distal side Asp 152 [68] and Asn 153 [69] of SyKatG were also found to be critical for catalatic activity, but not for peroxidatic activity. These two residues are conserved in all known catalase-peroxidase sequences except that of Salmonella typhimurium [70], where the Asn is replaced by a Thr.…”

Probing the structure and bifunctionality of catalase-peroxidase (KatG)

“…In order to gain insight into the structure-function properties of KatGs, variants have been produced for KatGs from E. coli [24], M. tuberculosis [8,13,[17][18][19][20][30][31][32][33][34]38], Synechocystis [7,12,[14][15][16][21][22][23][25][26][27][28][29]35] and B. pseudomallei [36,39,40]. Very similar results were obtained irrespective of the origin of the KatG mutated.…”

“…At first glance KatG seems to behave in a similar manner. Mixing of native KatG (0.5-2 lM) with a 10-100-fold excess of H 2 O 2 only slightly perturbed the absorption spectrum from that of the resting enzyme [21][22][23][24][25][26][27]30]. Initially, in analogy to monofunctional catalases, this was attributed to the fact that the rate of Compound I reduction significantly exceeds that of its formation.…”

“…9a) [7,8,[21][22][23][24][25][26][27]30]. The combined use of multifrequency (9-285 GHz) EPR spectroscopy, isotopic labeling and site-directed mutagenesis, has shown that Synechocystis wild-type KatG forms the oxoiron(IV) porphyrin radical intermediate followed by a tryptophanyl radical and a tyrosyl radical when the ferric protein was mixed with PAA (Reaction 3) [28].…”

Role of the Oxyferrous Heme Intermediate and Distal Side Adduct Radical in the Catalase Activity of Mycobacterium tuberculosis KatG Revealed by the W107F Mutant

“…More recently, residue Trp 111 together with His 112 were described as key sites in Synechocystis KatG for the steering of Cmpd I reduction by hydrogen peroxide (16). Mutations in other residues surrounding the heme pocket have also been shown to interfere with catalase activity for reasons ascribed to disruption of hydrogen bonding networks (17,18). Other issues such as stabilization of the oxyferrous heme intermediate formed in the presence of excess H 2 O 2 was not presented in studies of KatG from other laboratories, but in the case of M. tuberculosis KatG[Y229F] (14) and in KatG[W107F] as shown here, such stabilization is clearly a fundamental alteration in function that sets apart the behavior of these mutants.…”