Structural basis for organism-dependent effects on spectroscopic properties of type 1 copper azurin by the same axial methionine to histidine mutation

Abstract

Cupredoxins are a family of copper proteins that mediate electron transfer in key biological processes, and understanding structural features responsible for their spectroscopic properties is important for elucidating structure–function relationships. Here, we characterize the effect of mutation of the axial methionine to histidine in azurin from Pseudomonas aeruginosa (M121HAz-PA) using UV–Vis absorption, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography to examine how the same M121H mutation affects spectroscopic properties differently from its homolog in Alcaligenes denitrificans (M121HAz-AD). M121HAz-PA shows a pH-dependent UV–vis spectral change from pH 4 to pH 8, with unusual EPR features, indicating changes in coordination electronic structure. Azide binding titrations produce EPR spectra intermediate between type 1 copper (T1Cu) and type 2 copper (T2Cu), supporting that changes of (exogenous) ligand coordination drive significant geometric and electronic rearrangements. X-ray crystallography reveals differences in axial His121 coordination mode to Cu^II and its orientation between M121HAz-PA and M121HAz-AD, providing a structural basis for the observed spectroscopic differences. These findings provide insights into how subtle variations in ligand coordination affect the electronic structure in Cu-binding sites, offering a basis for the rational design of metalloproteins.