in the absence of substrate. Thus, also in the absence of externally added H2O2, trace amounts of H2O2 generated by LPMOs may fuel the catalytic reaction. Furthermore, experimental studies are affected by possible non-enzymatic reactions involving reducing agents, free copper including copper leaked from denatured LPMOs, and added or in situ produced H2O2, further complicating the analysis of experimental results. These complications have contributed to a situation where the details regarding the LPMOs cosubstrate (O2 or H2O2) are still controversial. Regardless of whether LPMOs react according to Scheme (1) as monooxygenases or according to Scheme (2) as peroxygenases, or variations thereof, it has been firmly demonstrated that an initial reduction of the Cu(II) resting states is required for catalytic turnover (see reactions 1a and 1b in Figure ). This is also true when LPMOs react as oxidases without substrate and produce H2O2 according to Scheme (3). This initial reduction has been denoted a "priming" reduction, for which a variety of reductants can be employed. It has been shown that both the Cu(I) state and the Cu(II) resting state can bind the substrate (according to reactions 2a and 2b in Figure ), but some experimental studies show that the reduced LPMO has a 2 to 10-fold higher affinity for cellulose. Therefore, it has been proposed that metal reduction precedes substrate binding. The nature of the priming reduction separates LPMOs from other metalloenzymes that also activate inactive C-H bondsbut whose reaction cycle otherwise shows similarities to the LPMOs' catalytic cycle 41 : The iron-heme enzyme cytochrome P450 usually forms a highly oxidizing species (Cpd1) from O2. The resting state Fe(III)-heme unit is first reduced after substrate binding, and O2 binds to this Fe(II)-heme unit (similar to the O2 pathway with reactions 3b and 4b in Figure ). The Fe(III)-O2 -species formed from initial O2 binding is further reduced and protonated into a Fe(III)-OOH intermediate (Cpd0). The HOO -unit in Cpd0 is heterolytically cleaved with help from nearby residues that can participate in acid/base chemistry under oxidation of Fe(III) to the oxidative species (Cpd1), responsible for C-H activation. Thus, cytochrome P450 needs a continuous flow of electrons in its catalytic cycle. This is different from LPMOs, which can perform up to 20 catalytic cycles after one priming electron has been added (when they work as peroxygenases). The iron-heme unit is also used by peroxidases, such as cytochrome c peroxidase and horseradish peroxidase (HRP). Unlike most cytochrome P450s, the peroxidases form the oxidative species (Cpd1) from H2O2 binding to the ferric Fe(III)-heme resting state. Thus, this is similar to the H2O2 pathway with reactions 5a/5b and 6a/6b in Figure , with the important difference that it is the oxidized form of the heme unit that interacts with H2O2, and no reduction is required for H2O2 activation. Instead, the binding of H2O2 generates the Fe(III)-OOH intermediate (after deprotonation of H2O2). The P450 cytochromes can use a similar mechanism known as the "peroxide shunt" pathway, but this generally leads to a one-electron reduced form of Cpd1, denoted Cpd2, and a protein radical .