Redox equilibria of manganese peroxidase from Phanerochaetes chrysosporium: functional role of residues on the proximal side of the haem pocket

Abstract

Redox potentials of recombinant manganese peroxidase from Phanerochaetes chrysosporium have been measured by cyclic voltammetry as a function of pH, between pH 4.5 and pH 10.5. They display a bimodal behaviour (characterized by an ‘alkaline’ and an ‘acid’ transition), which indicates that (at least) two protonating groups change their pKb values upon reduction (and/or oxidation) of the iron atom in haem. Analogous measurements have been carried out on four site-directed mutants involving residues in close proximity to the proximal ligand, His173, in order to investigate the role played by residues of the proximal haem pocket on the redox properties of this enzyme. Results obtained suggest that the protonation state of N∆ of the proximal imidazole group is redox-linked and that it is crucial in regulating the ‘alkaline’ transition. On the other hand, none of the proximal mutants alters the ‘acid’ transition, suggesting that it is modulated by groups located in a different portion of the protein.

Characterization of Dye-decolorizing Peroxidase (DyP) from Thermomonospora curvata Reveals Unique Catalytic Properties of A-type DyPs

“…Indeed, when the titration was performed at pH 6, the observed n app (0.79) was much closer to the theoretical value. Moreover, the E°Ј of TcDyP was strongly influenced by pH, which is consistent with other peroxidases (52)(53)(54). One pH unit corresponded to a ⌬E°Ј of ϳ60 mV for wt-TcDyP.…”

Increase of Redox Potential during the Evolution of Enzymes Degrading Recalcitrant Lignin

“…[17] The reduction potentials of the three redox couples in extant/ancestral fungall igninolytic peroxidases are highert han those reported for most animal, [27] plant [34] or prokaryotic [30] peroxidases, usually estimated atp H7.H owever,t he differences with the E8' (CI/RS) values of some animal peroxidases, [27] which were estimated with as imilar stopped-flow method, would be small after considering the variation (around 0.2 V) existing between the pH 7a nd pH 3e stimations, [22,35,36] due to the presence of one or several protonable residues contributing to peroxidase catalysis. [25,37] The values shown here are also slightly higher than the E8' of 1.3 Ve stimated at pH 3, with the stopped-flow method,f or the CI/RS couple of fungal chloroperoxidase and peroxygenase, [36] two heme-thiolate proteins related to cytochromeP 450 monooxygenases.…”

“…The most common redox measurement in heme proteins, basidiomycete peroxidases included, [20][21][22][23][24][25] is the midpoint po-tentialo ft he ferric/ferrous transition ( Figures S3 and S4,S upporting Information), even thoughi ti sn ot part of the catalytic cycle (Figure2). To explore the mechanistic implicationso fr eductionp otentiali nt he peroxidasec atalytic cycle, we used stopped-flow spectrophotometry [26][27][28][29] to measuret he concentration of the oxidized and reduced forms of the different enzymes in their RS/CI and CII/RS transitions ( Figures S5 and S6, respectively) as illustrated in Figure 3f or the most recent ancestor.S topped flow was used here to assign the equilibrium concentrations of the two redox states of the enzyme and the two redox states of the substrates used, providing an equilibrium constant that, with the use of the Nerst equation, will allow for the determination of the midpoint potential.…”

The prospects for peroxidase-based biorefining of petroleum fuels

“…However, mixed results have been observed. In CcP, substitution with a Glu residue led to an increase of 70 mV in the redox potential (Goodin & McRee 1993), whereas in MnP, the same substitution resulted in a decreased redox potential (Santucci et al 2000). Moreover, substitution with the poor hydrogen-bonding Asn in CiP decreased the redox potential of the enzyme, which is in clear contradiction with the hypothesis mentioned above (Ciaccio et al 2003).…”