New Insights into the Heme Cavity Structure of Catalase-Peroxidase: A Spectroscopic Approach to the Recombinant Synechocystis Enzyme and Selected Distal Cavity Mutants

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Abstract
Catalase-peroxidases (KatGs) are heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs exhibit a peroxidase activity of broad specificity and a high catalase activity, which strongly depends on the presence of a distal Trp as part of the conserved amino acid triad Arg-Trp-His. By contrast, both CCP and APX do not have a substantial catalase activity despite the presence of the same triad. Thus, to elucidate structure−function relationships of catalase-peroxidases (for which no crystal structure is available at the moment), we performed UV−Vis and resonance Raman studies of recombinant wild-type KatG from the cyanobacterium Synechocystis PCC 6803 and the distal side variants (His123→Gln, Glu; Arg119→Ala, Asn; Trp122→Phe, Ala). The distal cavity of KatG is very similar to that of the other class I peroxidases. A H-bond network involving water molecules and the distal Trp, Arg, and His is present, which connects the distal and proximal sides of the heme pocket. However, distal mutation not only affects the heme Fe coordination state and perturbs the proximal Fe−Im bond, as previously observed for other peroxidases, but also alters the stability of the heme architecture. The charge of the distal residues appears particularly important for maintaining the heme architecture. Moreover, the Trp plays a significant role in the distal H-bonding, much more pronounced than in CCP. The relevance of these findings for the catalase activity of KatG is discussed in light of the complete loss of catalase activity in the distal Trp mutants.