Understanding the initial events of the oxidative damage and protection mechanisms of the AA9 lytic polysaccharide monooxygenase family

Lytic polysaccharide monooxygenase (LPMO) is a new class of oxidoreductases that boosts polysaccharide degradation employing a copper active site. This boost may facilitate the cost-efficient production of biofuels and high-value chemicals from polysaccharides such as lignocellulose. Unfortunately, self-oxidation of the active site inactivates LPMOs. Other oxidoreductases employ hole-hopping mechanisms as protection against oxidative damage, but little is generally known about the details of these mechanisms. Herein, we employ highly accurate theoretical models based on density functional theory (DFT) molecular mechanics (MM) hybrids to understand the initial steps in LPMOs' protective measures against self-oxidation; we identify several intermediates recently proposed from experiment, and quantify which are important for protective hole-hopping pathways. Investigations on two different LPMOs show consistently that a tyrosine residue close to copper is crucial for protection: this explains recent experiments, showing that LPMOs without this tyrosine are more susceptible to self-oxidation.

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2024 Energies and distances of all states associated with reaction I: Table S1: QM/MM energies (in kJ/mol) for TaAA9 and LsAA9 for reaction I in reference to the respective reactant [CuO] + (1) in the triplet state.Energies were obtained with def2-TZVPP based on structures optimized with TPSS/def2-SV(P).a TS I* and Int I were only observed for the triplet state.

Energy difference
Table S2: Atom distances for chosen atoms in Å for the most stable electron configurations for reaction I. Structures were optimized using TPSS/def2-SV(P).are rather functional dependent for both isomers.While B3LYP shows small splittings of 0.2-8.9kJ/mol, TPSS predicts larger splittings of 19.9-20.6 kJ/mol depending on the isomer.
Both isomers only show small changes for the spin densities and are therefore expected to have a similar electronic structure.Energies and distances of all states associated with reaction II: a Calculations collapse into closed-shell singlets close to the TS (here, last proper open-shell singlet energies are given as TS II for S = 0).

UV-vis spectra
Figure S5: Calculated UV-vis spectra of 2 for TaAA9 in the triplet spin state.The spectra were obtained using CAM-B3LYP/def2-TZVPP in vacuum or with point charges (the same charges as used in the QM/MM calculations).The spectra with point charges was calculated for the untruncated QM region as shown in Fig. 2. orbitals of hydroxyl lone pair character to orbitals of π-character on tyrosine.We note that the spectrum of the tyrosyl isomer 2 ′ (not shown) of TaAA9 does not show this peak, while otherwise showing almost identical features to the spectrum of intermediate 2.Moreover, this transition is also not present if the spectrum is calculated with pointcharges (see Fig. S5).shows two less intense peaks at higher energies at 392 nm (3.16 eV) and 294 nm (4.22 eV) with a highly delocalized nature of the transitions.
Intermediate 3 for TaAA9 also shows the intense feature around 400 nm seen for 3 in the LsAA9 spectrum.However, an additional transition is also seen at higher energy: the two most intense peaks in Fig. S7 (green spectrum, left) are at 342 nm (3.63 eV) and 395 nm (3.14 eV).An analysis of the major orbital contributions of these peaks show a large involvement of the histidyl.The main peak at 395 nm mainly involves transitions from orbitals of hydroxyl lone pair character to an orbital of π-character on the histidyl His1.The less intense peak at higher energies (342 nm) occurs due to ligand-to-metal charge transfer transitions including orbitals on the histidyl.Similar transitions can also be observed for the isomer 3 ′ , albeit with the two most intense peaks red-shifted to 348 nm (3.57eV) and 406 nm (3.05 eV) for the triplet state and 384 nm (3.23 eV) and 416 nm (2.98 eV) for the open-shell singlet.Overall, the spectra of 3 and 3 ′ are quite similar.
Fig.4c) the hydrogen bond between the O−H Tyr175 and the secondsphere Gln173 is broken with a low activation energy of 29 kJ/mol.In a second step, the hydrogen of the tyrosine hydroxy group is transferred to O ox .It was possible to obtain a stable intermediate "Int I " (we could ony obtain this intermediate in the triplet state and it was not obtained for LsAA9).Since the energy of this intermediates is essentially the same as the transition state, we decided not to include it in Fig.3or Fig. 4 (this does not change any conclusions).The corresponding figures with "Int I " included are shown here, see Fig. S1 and S2.The latter Figure also includes a stable conformer of the tyrosine (2 ′ ), shown in Fig. S2, not shown in the main paper.

Figure S1 :
Figure S1: Energy diagram (in kJ/mol) for the reaction I for TaAA9.The reactant 1 in the triplet state was used as the reference.Energies were obtained employing def2-TZVPP based on structures optimized with TPSS/def2-SV(P).Results in bold style were obtained for the triplet state and those in italic were obtained for the open-shell singlet state.

Figure S2 :
Figure S2: The hydrogen-abstraction reaction I illustrated for TaAA9 (a-e).An alternative conformation (2 ′ ) of the tyrosyl 2 is shown in f.The structures were optimized using TPSS/def2-SV(P) while the energies shown are obtained employing B3LYP/def2-TZVPP.Only the structures for the most stable electron configuration (either open-shell singlet or triplet) is shown.Key distances are given in Å and energies in kJ/mol with reference to reactant 1 in the triplet state.

Figure S3 :
FigureS3: QM regions for TaAA9 (left) and LsAA9 (right) employed for the TD-DFT calculations (histidyl 3 as example).The structures were QM/MM optimized using TPSS/def2-SV(P).The TD-DFT structures were slightly truncated compared to the QM regions used for the QM/MM calculations (see computational details).Labels refer to PDB 2YET 22 and 5ACF 47 for TaAA9A and LsAA9, respectively.

Figure S7 :
Figure S7: Calculated UV-vis spectra for the histidyl in TaAA9.Comparison of 3 and 3 ′ in the triplet state (left) and comparison of the triplet state of 3 with the open-shell singlet state of 3 ′ (right).The spectra were obtained using CAM-B3LYP/def2-TZVPP.The structures were obtained from the QM/MM calculations employing TPSS/def2-SV(P) with the smaller QM region not including Phe43.
Distance Intermediate Cu-O ox Cu-N δ Additional discussion of reaction I: It should be noted that in the first part of the reaction for TaAA9 (

Table S3 :
QM/MM energies for TaAA9 and LsAA9 in reference to the reactant [CuO] + (1) in the triplet state.Energies for TaAA9 were obtained with def2-TZVPP based on structures optimized with TPSS/def2-SV(P).Energies for LsAA9 were taken from ref.35.

Table S5 :
Energies and distances of all states associated with reaction III: QM/MM energies (in kJ/mol) calculated with the smaller QM region for TaAA9 and LsAA9 for reaction III.The energies are in reference to the respective reactant in the triplet state (3 ′ and 3 for TaAA9 and LsAA9, respectively).Energies were obtained with def2-TZVPP based on structures optimized with TPSS/def2-SV(P).All energies for the larger QM regions can be obtained directly from Fig.7of the main paper.
a Energies include high MM energies (>33 kJ/mol) b Reaction ended in spontaneous rebound of the O hyd group from the Cu with the histidyl

Table S6 :
Atom distances for chosen atoms in Å for reaction III.The structures were optimized with the bigger QM region (including Phe43 and Pro30 for TaAA9 and LsAA9, respectively) employing TPSS/def2-SV(P).