Methionine Ligand Interaction in a Blue Copper Protein Characterized by Site-Selective Infrared Spectroscopy

Abstract

The reactivity of metal sites in proteins is tuned by protein-based ligands. For example, in blue copper proteins such as plastocyanin (Pc), the structure imparts a highly elongated bond between the Cu and a methionine (Met) axial ligand to modulate its redox properties. Despite extensive study, a complete understanding of the contribution of the protein to redox activity is challenged by experimentally accessing both redox states of metalloproteins. Using infrared (IR) spectroscopy in combination with site-selective labeling with carbon-deuterium (C-D) vibrational probes, we characterized the localized changes at the Cu ligand Met97 in the oxidized and reduced states, as well as the Zn(II) or Co(II)-substituted, the pH-induced low-coordinate, the apoprotein, and the unfolded states. The IR absorptions of (d3-methyl)Met97 are highly sensitive to interaction of the sulfur-based orbitals with the metal center and are demonstrated to be useful reporters of its modulation in the different states. Unrestricted Kohn-Sham density functional theory calculations performed on a model of the Cu site of Pc confirm the observed dependence. IR spectroscopy was then applied to characterize the impact of binding to the physiological redox partner cytochrome (cyt) f. The spectral changes suggest a slightly stronger Cu-S(Met97) interaction in the complex with cyt f that has potential to modulate the electron transfer properties. Besides providing direct, molecular-level comparison of the oxidized and reduced states of Pc from the perspective of the axial Met ligand and evidence for perturbation of the Cu site properties by redox partner binding, this study demonstrates the localized spatial information afforded by IR spectroscopy of selectively incorporated C-D probes.

Site-Specific 1D and 2D IR Spectroscopy to Characterize the Conformations and Dynamics of Protein Molecular Recognition

“…19,[49][50][51][52][53] Residue-selective labeling with deuterated amino acids or analogues similar to natural amino acids, such as the methionine analogue azidohomoalanine (Aha), can be achieved through expression in defined media in an auxotrophic host or one that cannot efficiently biosynthesize the amino acid. 24,27,34,54 This approach is most useful for labeling rare amino acids, as they may be present at only one or a few positions, or in proteins that can tolerate the mutational removal of all but one instance of an amino acid. Unique residues also can be functionalized to generate site-specific IR probes through chemical post-translational modification.…”

“…C-D bonds are not only the least perturbative probes, but they also provide intrinsic probes of the protein itself. 19,[23][24][25][26][27] While in many ways they are our ideal probes, their small transition dipole strengths make their absorptions difficult to discern, and their use as 2D IR probes is not yet feasible. Nonetheless, we believe that their appeal makes their development worthy of further exploration.…”

“…To enable the use of IR spectroscopy to selectively investigate the metal site of Pc, we incorporated C-D probes at the methyl group of the axial Met97 ligand (d 3 Met97). 27 The C-D vibrations of d 3 Met97 were highly sensitive to the interaction of the adjacent sulfur-based orbitals with the positively charged metal center, making them useful reporters of the Cu-Met97 bond ( Figure 4B). Specifically, the symmetric stretch of d 3 Met97 showed a shift to higher frequency upon oxidation of Cu(I) Pc to Cu(II) Pc.…”

Azidohomoalanine: A Minimally Invasive, Versatile, and Sensitive Infrared Label in Proteins To Study Ligand Binding

“…[15][16][17][18][19] Such studies require a special vibrational label that absorbs in the spectral window between ≈1700 cm −1 to ≈2800 cm −1 in order to discriminate it from a huge protein background. [20][21][22][23][24][25] Among the possible molecular groups that have been proposed in that regard, 16,17,19,[26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] we favor the noncanonical amino acid azidohomoalanine (Aha). Aha has a relatively high extinction coefficient (≈300-400 M −1 cm −1 ), 15 and it has been shown to be an environment-sensitive infrared probe, capable of sensing the polarity of its environment via the frequency of its vibrational transition 15,18,28,[45][46][47] Additionally, this noncanonical amino acid can be incorporated into a protein in virtually any position via methionine-auxotrophic expression strategy.…”

Heterogeneous and Highly Dynamic Interface in Plastocyanin–Cytochrome f Complex Revealed by Site-Specific 2D-IR Spectroscopy

“…Two-dimensional (2D) IR spectroscopy enables the measurement of the inhomogeneity of the probe frequencies, which affords a more rigorous measure of local heterogeneity. 31,32 Moreover, time-dependent 2D-IR experiments can follow in real time the dynamics among the frequency distribution (spectral diffusion), providing a direct measure of the dynamics of the interaction of the probe with its environment. [33][34][35][36][37][38][39] We applied 1D and 2D spectroscopy in combination with site-selective labeling with IR probes toward better understanding the complexes formed between Pc and cyt f from Nostoc cyanobacteria.…”