UV Raman monitoring of histidine protonation and H-²H exchange in plastocyanin

UV resonance Raman bands of Cu-bound and protonated histidine residues have been detected in ²H₂O solutions of poplar plastocyanin. For the Cu(II) protein, slow NH-²H exchange of the His37 ligand was monitored via the growth of bands at 1389 and 1344 cm^-1 when Pcy was exchanged into ²H₂O, or via their diminution when the protein was exchanged back into H₂O; the rate constant is 7×10^-4/s at pH (p²H) 7.4 at room temperature. The slow exchange is attributed to imidazole H-bonding to a backbone carbonyl. Nearby bands at 1397 and 1354 cm^-1, appear and disappear within the mixing time, and are assigned to the solvent-exposed His87 ligand. The ∼10 cm^-1 differences between His37 and His87 are attributed to the effect of H-bonding on the imidazole ring modes. The UVRR spectra of the Cu(I) protein in ²H₂O reveal a 1408 cm^-1 band, characteristic of NH-²H-exchanged histidinium, which grows in as the p²H is lowered. Its intensity follows a titration curve with pK_a=4.6. This protonation is assigned to the His87 residue, whose bond to the Cu(I) is known from crystallography to be broken at low pH. As the 1408 cm^-1 band grows, a band at 1345 cm^-1 diminishes, while another, at 1337 cm^-1 stays constant. These are assigned to modes of bound His87 and His37, respectively, shifted down 7-9 cm^-1 from their Cu(II) positions.