Electrospray ionization (ESI) mass spectrometry (MS) is a crucial method for rapidly determining the interactions between small molecules and proteins with ultrahigh sensitivity. However, nonvolatile molecules and salts that are often necessary to stabilize the native structures of protein–ligand complexes can readily adduct to protein ions, broaden spectral peaks, and lower signal-to-noise ratios in native MS. ESI emitters with narrow tip diameters (∼250 nm) were used to significantly reduce the extent of adduction of salt and nonvolatile molecules to protein complexes to more accurately measure ligand–protein binding constants than by use of conventional larger-bore emitters under these conditions. As a result of decreased salt adduction, peaks corresponding to protein–ligand complexes that differ in relative molecular weight by as low as 0.06% can be readily resolved. For low-molecular-weight anion ligands formed from sodium salts, anion-bound and unbound protein ions that differ in relative mass by 0.2% were completely baseline resolved using nanoscale emitters, which was not possible under these conditions using conventional emitters. Owing to the improved spectral resolution obtained using narrow-bore emitters and an analytically derived equation, K d values were simultaneously obtained for at least six ligands to a single druggable protein target from one spectrum for the first time. This research suggests that ligand–protein binding constants can be directly and accurately measured from solutions with high concentrations of nonvolatile buffers and salts by native MS.

Cytochrome P450 heme-thiolate monooxygenases are exceptionally versatile enzymes which insert an oxygen atom into the unreactive C–H bonds of organic molecules. They source O2 from the atmosphere and usually derive electrons from nicotinamide cofactors via electron transfer proteins. The requirement for an expensive nicotinamide adenine dinucleotide (phosphate) cofactor and the redox protein partners can be bypassed by driving the catalysis using hydrogen peroxide (H2O2). We demonstrate that the mutation of a highly conserved threonine residue, involved in dioxygen activation, to a glutamate shuts down monooxygenase activity in a P450 enzyme and converts it into a peroxygenase. The reason for this switch in the threonine to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas palustris HaA2 was linked to the lack of a spin state change upon the addition of the substrate. The crystal structure of the substrate-bound form of this mutant highlighted a modified oxygen-binding groove in the I-helix and the retention of the iron-bound aqua ligand. This ligand interacts with the glutamate residue, which favors its retention. Electron paramagnetic resonance confirmed that the ferric heme aqua ligand of the mutant substrate-bound complex had altered characteristics compared to a standard ferric heme aqua complex. Significant improvements in peroxygenase activity were demonstrated for the oxidative demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and veratric acid to vanillic acid (up to 6-fold). The detailed characterization of this engineered heme peroxygenase will facilitate the development of new methods for driving the biocatalytic generation of oxygenated organic molecules via selective C–H bond activation using heme enzymes.

Cytochrome P450 CYP199A4 from Rhodopseudomonas palustris catalyses heteroatom dealkylations, sulfoxidation and amide and cyclic hemiacetal formation

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