A Role for Catalase-Peroxidase Large Loop 2 Revealed by Deletion Mutagenesis: Control of Active Site Water and Ferric Enzyme Reactivity

Abstract

Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

Protein-Derived Cofactors Revisited: Empowering Amino Acid Residues with New Functions

“…108 Furthermore, the structure of KatG contains a large loop, not present in the peroxidase, which is essential for catalase activity. 109, 110 It is believed that heme is required to generate the cross-linked amino acids during the maturation of the active site. 111 The cross-linked amino acids in addition to the heme are then for its catalatic activity.…”

“…However, the Met-Tyr-Trp cross-link is only seen in KatG, and site-directed mutagenesis studies demonstrated that the cross-link is needed for the catalatic activity, but not for peroxidatic activity . Furthermore, the structure of KatG contains a large loop, not present in the peroxidase, which is essential for catalase activity. , It is believed that heme is required to generate the cross-linked amino acids during the maturation of the active site . The cross-linked amino acids in addition to the heme are then required for its catalatic activity.…”

Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme

“…Among them are two large loops (LL1 and LL2), both of which are essential for KatG function (20 -22, 26, 27). Both loops also contribute to a much narrower channel to the active-site heme, which among other things restricts access of many peroxidatic electron donors (PxEDs) to the heme edge (22,(27)(28)(29). Additionally, LL1 bears an invariant tyrosine (Tyr-229 by MtKatG numbering) that participates in a unique methionine-tyrosine-tryptophan (MYW) adduct that serves as a protein-derived cofactor (20 -22, 30, 31).…”

Interaction with the Redox Cofactor MYW and Functional Role of a Mobile Arginine in Eukaryotic Catalase-Peroxidase

“… 13 , 14 Mutation of any of these KatG-typical structural features typically reduces the catalase activity without compromising the peroxidase activity. 13 − 22 …”

“…Similar to the prokaryotic counterparts, − eukaryotic KatGs have two interdependent cooperating redox-active cofactors at their active site, namely a heme b and a unique posttranslationally and autocatalytically formed Met299-Tyr273-Trp140 (MYW) adduct [extracellular Magnaporthe grisea KatG ( Mag KatG2) numbering throughout]. KatG can be described as a catalase in peroxidase clothing because it has been engineered by Nature to catalyze efficient H 2 O 2 dismutation similar to monofunctional catalases. , Together with cytochrome c peroxidases, ascorbate peroxidases and hybrid-type peroxidases KatGs comprise Family I of the peroxidase-catalase superfamily. , Besides the MYW adduct mentioned above, further KatG-specific structural peculiarities include (i) a two-domain structure with only the N-terminal domain containing the two redox cofactors and essential catalytic residues, ,, (ii) loop insertions that contribute to the architecture of the long and restricted access channel, and (iii) a fully conserved aspartate at the entrance of the substrate channel to the heme cavity. , Mutation of any of these KatG-typical structural features typically reduces the catalase activity without compromising the peroxidase activity. − …”