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

Qiang Wu, Fangbiao Li, Weixun Wang, Michael H. Hecht, Thomas G. Spiro

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

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 pKa=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.

Original languageEnglish (US)
Pages (from-to)381-387
Number of pages7
JournalJournal of Inorganic Biochemistry
Volume88
Issue number3-4
DOIs
StatePublished - Mar 20 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Inorganic Chemistry

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