Accumulation of H2O2 when incubating NcLPMO9C with reductant in the absence and presence of substrate. The enzyme (0.87 μM), which was the first LPMO for which activity on oligomeric substrates was shown, was incubated with 30 μM ascorbic acid, the reactants of the Amplex red assay and 5 mM of the indicated potential substrate, at pH 6.0 . Ref, no substrate added. Control experiments without the reductant or the LPMO did not show H2O2 accumulation. Please note that the lower levels of H2O2 in reaction mixtures that contain substrates that are cleaved by the enzyme (Glc5 and Glc6) do not necessarily indicate that H2O2 was not produced, as was thought at the time; it is also possible that H2O2 was indeed produced but did not accumulate to the same extent because it was used by the LPMO when cleaving the substrate; see text for more details.