Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage

Abstract

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide–dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

Biochemical features of dye‐decolorizing peroxidases: Current impact on lignin degradation

“…The conserved arginine in the active site was also studied. The role of this residue seems to be structural as its mutation compromises the architecture of its distal heme cavity and access channel [72].…”

“…The two domains, alpha and beta, form the active site cavity that hosts heme. To date, 39 crystal structures of DyPs are known (Table 1) [64,70,71,72,75,[77][78][79][80][81][82][83][84][85][86][87][88][89][90]. Detailed studies of the catalytic cycle show that hydrogen peroxide deprotonation is the first step (Fig.…”

“…At this point, heterolytic cleavage of the O-O can occur leading to the formation of the Fe(IV)-O porphyrin radical Compound I. The formation of Compound II (FeIV-OH) from Compound I is achieved through a one-electron reduction producing a first radical species [72]. Finally, the ferric iron resting state (FeIII) is regenerated by a second one-electron reduction with generation of a second radical [72].…”

“…The formation of Compound II (FeIV-OH) from Compound I is achieved through a one-electron reduction producing a first radical species [72]. Finally, the ferric iron resting state (FeIII) is regenerated by a second one-electron reduction with generation of a second radical [72]. An earlier shunt of the catalytic cycle is also possible through immediate two-electron reduction of Compound I generating ferric iron resting state (FeIII).…”

Rational design of heme enzymes for biodegradation of pollutants toward a green future

“…Instead of the common distal Arg-His pair in peroxidases, DyPs have a pair of Arg-Asp in the heme distal site (Fig. 1A) [45], which acts as an acid-base catalyst and plays crucial roles in heterolytic cleavage of H 2 O 2 [40,46]. Moreover, multiple Tyr and Trp residues were revealed in the protein scaffold of DyPs.…”