Fungal decomposition of mineral-associated proteins through Fenton-based oxidation and enzymatic hydrolysis

A substantial fraction of nitrogen (N) in forest soils is present in mineral-associated proteinaceous compounds. The strong association between proteins and soil minerals protects these compounds from decomposition; however, previous studies have shown that ectomycorrhizal (ECM) fungi can acquire N via extracellular proteolytic enzymes acting on iron oxide mineral-associated proteins. Hydrolysis is accompanied by reductive dissolution of the iron oxides, creating conditions for Fenton chemistry and hence, generation of highly reactive hydroxyl radicals (HO^•). Yet, the specific mechanisms employed by ECM fungi to acquire N from these mineral-associated proteinaceous compounds remain largely unresolved. In situ IR spectroscopy was used to monitor the molecular-scale reactions of bovine serum albumin (BSA, as a model protein) with proteases and HO^• occurring at iron mineral interfaces. The decomposition of ferrihydrite-associated BSA by the ECM fungus Suillus luteus was followed using optical photothermal infrared (O-PTIR) microspectroscopy at the individual hyphal level. The effects and interplay between the oxidative and hydrolytic mechanisms in degrading and liberating N from mineral-associated BSA were examined using in vitro experiments. Proteolysis and oxidative mechanisms generated distinct, diagnostic IR spectral fingerprints of the mineral-adsorbed BSA. By correlating IR fingerprints with microspectroscopy of the fungal extracellular polymeric substance (EPS) region, we show that S. luteus decomposes mineral-associated proteins through sequentially deployed oxidative and hydrolytic mechanisms. BSA adsorbed on ferrihydrite is susceptible to HO^• generated in heterogeneous Fenton reactions, and carboxylates (e.g., oxalate) were generated that occupied adsorption sites on ferrihydrite, which can counteract the suppression of protease activity due to protease adsorption onto the mineral. Moreover, deamination and fragmentation were also observed during the Fenton reaction. Our findings underscore the previously overlooked role of extracellular oxidative chemistry in fungal acquisition of nitrogen from mineral-organic complexes.