X-ray Spectroscopy to Study Lytic Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenase (LPMO) is a family of mono-copper metalloenzymes that can perform oxidative saccharification of naturally abundant polysaccharides. The LPMO mechanism is of great interest because of the possible applications in the production of biofuels and in crop protection, however this mechanism remains largely uncharacterised.

This work aims to investigate the LPMO electronic structure over three key genomic families: AA9, AA10 and AA11, using conventional XAS and HERFD-XANES. The X-ray spectroscopy data presented is supported by EPR, UV-vis spectroscopy and TD-DFT calculations.

Changes to the electronic structure of the copper ion on the addition of substrate in both the Cu(I) and Cu(II) oxidation states have been evaluated. The main finding of this work is that the addition of PASC to Cu(II) AnAA9 does not yield any significant changes in the active site, whereas in the Cu(I) oxidation state the addition of phosphoric acid swollen cellulose induced changes consistent with a decrease in coordination number, which were observed spectroscopically and computationally. ICP-MS was used to detect the presence of metal contaminants in commonly used LPMO substrates, which found significant quantities of iron and zinc.

A Cu(II)-histidyl radical species has been observed and characterised. The radical intermediate was trapped for spectroscopic measurements using an anaerobic rapid freeze quenching technique. The Cu(II)-histidyl radical species has been found to be part of an LPMO oxidative damage protection mechanism.

Insights into the photodamage/ photoreaction mechanism of LPMOs were gained in the presence and absence of substrate. The spectra of intermediates were extracted using Multivariate Curve Resolution - Alternating Least Squares and linear combination fitting. It was found that there are discrepancies between chemically reduced and photoproduced LPMO samples and the most observed photodamage mechanisms were found to be specific primary and secondary reactions.

The main findings include direct comparisons of Cu(I) and Cu(II) LPMO-substrate interactions, the characterisation of a Cu(II)-histidyl intermediate species thought to be part of an oxidative damage protection mechanism, and a comprehensive investigation into X-ray radiation damage of LPMOs in the presence and absence of substrate.