Relationships of Ligand Binding, Redox Properties, and Protonation in Coprinus cinereus Peroxidase

Abstract

The pH dependence of the redox potentials and kinetics for CO association and dissociation was determined between pH 3.0 and 13.0 at 25°C for the wild-type Coprinus cinereus fungal peroxidase and for a site-directed mutant in which Asp 245, which is H-bonded to N ␦ of the imidazole of the proximal His 183, was substituted with Asn. The determination of these functional properties allowed this information to be merged in a self-consistent fashion and to formulate for the first time a complete scheme employing the minimum number of groups required to describe the whole proton-linked behavior of both redox and ligand binding properties. The overall pH dependence can be accounted for by four redox-and ligand-linked groups. The proximal H-bond, which is strictly conserved in all peroxidases, will still be present in the site-specific mutant, but will no longer have an ionic character, and this event will bring about an alteration of redox equilibria and CO binding kinetics, envisaging a relevant role played by this H-bond also in modulating redox properties and ligand binding equilibria.

pH-dependent redox and CO binding properties of chelated protoheme-l-histidine and protoheme-glycyl-l-histidine complexes

“…Such a feature can be correlated to the interrelationship in hemoproteins between the conformation of the axial coordination bond and the strain applied on the peptide associated with the coordinating residue. This phenomenon is observed in several hemoproteins, the most striking example being that of peroxidases, where a functional interrelationship has been documented between the proximal histidine and some residues on the proximal side of the heme pocket [48,49]. Furthermore, a very relevant finding of this investigation is indeed represented by the unraveling of a continuum of energy communication between the two sides of the heme, such as the influence of the strain on the proximal side of the heme is transmitted to the protonation of the H 2 O Scheme 3 Thermodynamic and kinetic relationships between different equilibria in heme-GH molecule on the distal side of the heme (Schemes 2, 3), since it is demonstrated that the effect does not require the assistance of a protein matrix.…”

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

“…The most common redox measurement in heme proteins, basidiomycete peroxidases included, is the midpoint potential of the ferric/ferrous transition (Figures S3 and S4, Supporting Information), even though it is not part of the catalytic cycle (Figure ). To explore the mechanistic implications of reduction potential in the peroxidase catalytic cycle, we used stopped‐flow spectrophotometry to measure the 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 for the most recent ancestor.…”

“…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

“…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). Clearly, the modulation of the redox potential in peroxidases is multifactorial and there are probably other contributing factors.…”