Altered coordination in a blue copper protein upon association with redox partner revealed by carbon–deuterium vibrational probes

Abstract

Proteins tune the reactivity of metal sites; less understood is the impact of association with a redox partner. We demonstrate the utility of carbon-deuterium labels for selective analysis of delicate...

The structure of plastocyanin tunes the midpoint potential by restricting axial ligation of the reduced copper ion

“…The FT IR spectra of d 3 Met97/ d 2 Cys89 for freshly prepared oxidized wt Pc and chemically reduced protein show absorptions in the transparent frequency window, as previously characterized 37 , 38 . Our analysis focused on the absorptions of the d 2 Cys89 CD 2 asymmetric stretch and d 3 Met97 CD 3 symmetric stretch modes due to their greater intensity (Supplementary Data 3 ).…”

“…No corresponding changes are found for d 2 Cys89 of the reduced protein. Based on our prior analysis 37 , 38 , the higher C-D frequencies found for the mutated Pc are indicative of stronger interaction between the metal ion and Met97. This relationship is illustrated, for example, by the higher C-D frequency of d 3 Met97 when interacting with the more highly charged Cu(II) versus the Cu(I) ion (Table 1 ).…”

“…In prior studies, we found the frequency of C-D vibrations of ligand d 2 Cys89 particularly sensitive to interaction with the metal ion. The frequency shifts by 26 cm −1 between the redox states and varies by as much as 45 cm −1 from the individual, reduced Pc to the oxidized Pc in complex with the redox partner cytochrome f 38 . C-D probes at Met97 are also modestly sensitive to changes at the metal site, differing by 3 cm −1 between redox states and up to 5.5 cm −1 for metal-substituted Pc 37 .…”

“…C-D probes at Met97 are also modestly sensitive to changes at the metal site, differing by 3 cm −1 between redox states and up to 5.5 cm −1 for metal-substituted Pc 37 . We previously demonstrated that C-D vibrations at the Cys89 and Met97 ligands are impacted by redox state, partner binding, metal substitution, demetallation, and unfolding 37 , 38 . Nonetheless, the vibrations display little change among oxidized wt and S9A, N33A, or N34A Pc, arguing that the Cu(II) coordination by the Cys89 and Met97 ligands is not significantly perturbed.…”

“…They introduce no perturbation, which is essential for the study of delicate metal sites in proteins. We previously have shown that C-D vibrations at Cys89 and Met97 in Pc are highly sensitive to the interaction of the ligands with the Cu ion 37 , 38 .…”