Theoretical and Computational Chemistry

The Ionic Atmosphere Effect on the Absorption Spectrum of a Flavoprotein: A Reminder to Consider the Importance of Solution Ions



Ionizable residues and monoatomic ions in solution modulate enzyme catalysis and the structural stability of proteins; however, the delicate interplay between these short-range charges and long-range charges, and their contributions to the electrostatic environment in a protein active site, is currently not fully understood. The study presented here utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 (NQO, EC, UniProtKB Q9I4V0) as a model system to study the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. Using pH-dependent UV-visible spectroscopy, there were no observed changes in the flavin absorption spectrum when an active site tyrosine (Y277) deprotonated in vitro. These results could only be reproduced computationally using Average Solvent Electrostatic Configuration (ASEC) hybrid quantum mechanics / molecular mechanics (QM/MM) simulations that included both positive and negative solution ions. The same calculations performed with minimal ions to neutralize protein charges predicted that deprotonating Y277 would significantly affect the flavin absorption spectrum. Analyzing the distribution of solution ions from ASEC and radial distribution functions derived from molecular dynamics indicated that the solution ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Biochemical experiments were performed to support this hypothesis. This work highlights the importance of salt ions, which are sometimes overlooked, since they can contribute a non-uniform and anisotropic long-range potential to the electrostatic environment of an active site.


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Supplementary material

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Supporting Information for: The Ionic Atmosphere Effect on the Absorption Spectrum of a Flavoprotein: A Reminder to Consider the Importance of Solution Ions
QM/MM computed changes in flavin oscillator strengths and excitation wavelengths from protonated Y277 with 18 added Na+ and Cl- ions to unprotonated Y277 with the added solution ions removed from the simulation space (Table S1). The UV-visible absorption spectrum for NQO-WT in 50 mM piperidine, pH 11.5, in the presence and absence of 100 mM KCl or KBr (Fig. S1).