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

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.