Oxidation-reduction potentials and ionization states of extracellular peroxidases from the lignin-degrading fungus Phanerochaete chrysosporium

Abstract

The oxidation-reduction potentials of lignin peroxidase isozymes H1, H2, H8, and H10 as well as the Mn-dependent peroxidase isozymes H3 and H4 are reported. The potentiometric titrations involving the ferrous and ferric states of the enzyme had Nernst plots indicating single-electron transfer. The Em7 values of lignin peroxidase isozymes H1, H2, H8, and H10 are -142, -135, -137, and -127 mV versus standard hydrogen electrode, respectively. The Em7 values for the Mn-dependent peroxidase isozymes H3 and H4 are -88 and -93 mV versus standard hydrogen electrode, respectively. The midpoint potential of H1, H8, and H4 remained unchanged in the presence of their respective substrates, veratryl alcohol and Mn(II). The midpoint potential between the ferric and ferrous forms of isozymes H1 and H4 exhibited a pH-dependent change between pH 3.5 and pH 6.5. These results indicate that the reductive half-reaction of the enzymes is the following: ferric peroxidase + le- + H+----ferrous peroxidase. Above pH 6.5, the effect of pH on the midpoint potential is diminished and indicates that an ionization with an apparent pKa equal to approximately 6.6-6.7 occurs in the reduced form of the enzymes. A heme-linked ionization group in the ferrous form of the enzymes was confirmed by studying the effect of pH on the absorption spectra of isozymes H1 and H4. These spectrophotometric pH titration experiments confirmed the electrochemical results indicating pKa values of 6.59 and 6.69 for reduced isozymes H1 and H4, respectively. These results indicate the presence of a heme-linked ionization of an amino acid in the reduced form of the lignin peroxidase isozymes similar to that of other plant peroxidases.

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

“…∆E p increased (up to 140-150 mV for scan rates of 500 mV\s), indicating the occurrence of quasi-reversibility. Figure 2 shows the pH-dependence of the redox potential for wt MnP, and the data at pH 7n0 agree with those already reported [23]. The data clearly showed that its redox behaviour was functionally modulated by the protonation of (at least) two groups, whose pK b values were redox-linked.…”

“…( \A #)! ), and the concentration was determined using an absorption coefficient, ε l 127 mM −" :cm −" at 407 nm [23]. Modified PG electrodes were prepared as previously described [24].…”

Reversible alkaline inactivation of lignin peroxidase involves the release of both the distal and proximal site calcium ions and bishistidine co-ordination of the haem

“…One of the key factors in evaluating the technological feasibility of these environmentally acceptable bio-bleaching systems is the stability of the peroxidases, particularly at high pH. Unfortunately, LiP has an extremely narrow pH optimum (pH 2.7-3.0) with respect to the majority of its substrates, and rapidly converts into a denatured species at pH 7.5 [23], particularly at elevated temperatures [24,25].…”

Mechanisms of Degradation by White Rot Fungi

“…The lignin peroxidases are somewhat unique in that they have higher oxidation potentials (--1.35 V) than do most peroxidases (--0.8 V) (7). In this way these enzymes have a somewhat greater range of chemicals that they can oxidize; however this does not explain why so many chemicals are oxidized by these fungi.…”