# Electrochemical Insight into the Copper Redox Chemistry and H2O2 and O2 Reducing Capability of Two AA10 Lytic Polysaccharide Monooxygenases

## TL;DR

This paper uses electrochemistry to study copper chemistry and catalytic activity of two LPMO enzymes, revealing insights into their function and performance under different conditions.

## Contribution

The study introduces electrochemical methods to directly measure LPMO redox chemistry and catalytic activity, offering a novel approach compared to traditional assays.

## Key findings

- CfAA10 outperforms CjAA10B in H2O2 and O2 reduction across pH 5–7.
- Both LPMOs show similar affinity-coupled specificity constants for H2O2 and O2.
- pH changes affect redox signals, with protonation of a glutamate residue influencing activity.

## Abstract

Lytic polysaccharide monooxygenases ([L]­PMOs) are copper-containing
enzymes that catalyse cleavage of the glycosidic bond, a process central
to microbial biomass degradation. Here, we describe electrochemical
methods used to investigate the Cu2+/1+ redox chemistry
and the polysaccharide-free catalytic activity of two AA10 LPMOs: CjAA10B from Cellvibrio japonicus and CfAA10 from Cellulomonas fimi. Immobilisation of these enzymes on the surface of a graphite electrode
allows for direct electrochemical measurements of Cu2+/1+ redox cycling as well as the ability of both LPMOs to reduce H2O2 vs O2. These measurements can be
advantageous when compared to biological dye assays as they provide
direct kinetic measurements and allow for investigation over a wider
range of environmental conditions. Values of k
cat and K
M- are reported for H2O2 and O2 reduction by CjAA10B and CfAA10 from pH 5–7, with CfAA10 consistently outperforming CjAA10B.
Both enzymes perform faster catalysis with H2O2 but when comparing the affinity-coupled specificity constant (k
cat/K
M), the LPMOs
perform similarly with both H2O2 and O2, suggesting both substrates are viable. We also note an increase
in redox signals as pH is decreased that correlates with EPR data
suggesting a second species is formed <pH 5, postulated to occur
due to the protonation of a glutamate residue (pK
a ∼ 4.6). The increase in signal size with decreasing
pH that is seen for the non-catalytic Cu2+/1+ transition
is interpreted in light of an increasing proportion of electroactive
species at low pH; such a change in activity with pH is notably not
observed in the presence of substrate (H2O2 or
O2). This suggests that substrate binding modulates the
active site, disrupting the effect of protonation. These findings
establish electrochemistry as a powerful tool for probing LPMO activity.