Activity, protection, damage or the rock, paper, scissors game in lytic polysaccharide monooxygenases (LPMO)

Hole hopping in proteins is the transfer of electron holes, which may also be viewed as radicals, generated during catalysis through amino acid side chains, primarily tyrosine and tryptophan. It was proposed as a protective mechanism by Harry B. Gray and Jay Winkler in 2015¹, where they analyzed the whole protein structure database to conclude that tyrosine and tryptophan chains are readily positioned for hole hopping in oxidoreductases. Hole hopping seems to stem from the primordial protection against oxidative damage provided by single tyrosine or tryptophan amino acids in solution². Hole hopping has been also finely tuned in evolution to be part of fascinating mechanisms, such as the magnetic avian compass³. The holes generated during catalysis in oxidoreductases are powerful oxidants used for the controlled and specific oxidation of substrates (marked as (1) in the figure). But if catalysis fails or if there is no substrate in the active site, holes can oxidize important amino acids around the active site (2). This would lead to damage and complete inactivation of the enzyme, unless there are protective hole hopping routes to scavenge these oxidants, directing them to locations far away from the precious active site, to eventually be defused by reductants present in solution (3).

In Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO), we analyze in detail such routes in a fascinating family of mono-copper enzymes with outstanding industrial potential. LPMOs, discovered by Vaaje-Kolstad et al^4,5, are capable of selectively activating C-H bonds in the crystalline parts of chitin and cellulose, two of the most abundant biopolymers on Earth. Breaking down the crystallinity of such polymers makes them more accessible for hydrolases, leading to biomass degradation in nature and to increased saccharification yields for the biomass-refining industry. LPMOs are small proteins (around 200 amino acids) with a characteristic flat surface where the Cu-atom is displayed for catalysis. During catalysis with H₂O₂, a hydroxyl radical or Cu-oxyl are generated as intermediates, representing the first hole. If substrate is bound, these powerful oxidants will oxidize C-H bonds. In the absence of substrate, the Cu-coordinating amino acids are damaged⁶.

Using a bacterial chitin-active LPMO as model, AA10A from Serratia marcescens, we analyzed in detail how hole hopping routes participate in the protection of this important family of proteins. Moreover, we showed that hole hopping influences catalysis, because it sometimes may compete with the productive reaction, i.e., the oxidation of the substrate.

The first step was identifying the amino acid candidates that may participate in hole hopping. Visual inspection combined with sequence space analysis of the whole bacterial LPMO repertoire and Rosetta design allowed us to design a set of enzyme mutants with changes in key amino acids (see figure). These mutants were then analyzed in detail with state-of-the art analytical techniques, involving stopped-flow spectroscopy, electronic paramagnetic resonance (EPR), detection of activity using microsensors⁷ and detection of oxidized products derived from chitin.

Stopped-flow spectroscopy in the absence of substrate allowed the detection of amino acid radicals formed during the reaction with H₂O₂, namely tyrosyl and tryptophanyl radicals. The detection of these radicals means that the hydroxyl or Cu-oxyl generated by H₂O₂ is transferred to nearby aromatic amino acids, which is a probe of hole hopping. By comparing the wild type LPMO with the various mutants, we were able to identify the main chain of hole hopping events and the amino acids involved. Additionally, by measuring chitin oxidation in damaging and non-damaging conditions, we could correlate the importance of every amino acid involved in hole hopping for protection during the oxidation of real substrates. This allowed us to unambiguously identify a strictly conserved tryptophan as a key residue for protection in all bacterial LPMOs.

Moreover, one of the variants displayed striking properties. By mutating the second tryptophane in the hole hopping route to phenylalanine in order to block hole transfer, we saw a significant boost in the initial rate of chitin oxidation, followed by faster inactivation. A set of control experiments, including EPR to show that mutagenesis does not affect the electronic properties of the Cu-site, showed that the only change in the variant was a slowed-down hole hopping route. Accurate measurement of catalytic rated using a H₂O₂ microsensor showed that indeed, this enzyme variant was faster than the wild-type.

Taken together, our results show that bacterial LPMOs active on chitin have a conserved hole hopping route that traverses the enzyme and is used for protection. Although LPMOs active on cellulose display different configurations of the protein core, the tryptophan that is closest to the copper is strictly conserved, highlighting its importance. We also show how activity and protection are competing processes in oxidoreductases, which is the first experimental evidence for a previously hypothesized⁸ delicate balance between redox activity and redox stability in redox enzymes.

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