Hydrogen bonding of sulfur ligands in blue copper and iron-sulfur proteins: detection by resonance Raman spectroscopy

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Abstract
The resonance Raman spectrum of the blue copper protein azurin from Alcaligenes denitrificans exhibits nine vibrational modes between 330 and 460 cm-1, seven of which shift 0.4-3.0 cm-1 to lower energy after incubation of the protein in D2O. These deuterium-dependent shifts have been previously ascribed to exchangeable protons to imidazole ligans [Nestor, L., Larrabee, J. A., Woolery, G., Reinhammar, B., and Spiro, T. G. (1984) Biochemistry, 23, 1084] or to exchangeable protons on amide groups which are hydrogen bonded to the cysteine thiolate ligands (a feature to all blue copper proteins of known structure). In order to distinguish between these two possibilities, a systemic investigation of Fe2S2(Cys)4-containing proteins was undertaken. Extensive hydrogen bonding between sulfur ligands and the polypeptide backbone had been observed in the crystal structure of ferredoxin from Spirulina platensis. The resonance Raman spectrum of this protein is typical of a chloroplast-type ferrodoxin and exhibits deuterium-dependent shifts of -0.3 to -0.5 cm-1 in the Fe-S modes at 283, 367, and 394 cm-1 (assigned to the bridging sulfurs) and -0.6 to -0.8 cm-1 in the Fe-S modes at 328 and 341 cm-1 (assigned to the terminal cysteine thiolates). Considerably greater deuterium sensitivity is observed in the Raman spectra of spinach ferredoxin and bovine adrenodoxin, particularly for the symmetric vibration of the Fe2S2 moiety at .apprx. 390 cm-1. This feature decreases by 0.8 and 1.1 cm-1, respectively, for the two oxidized proteins in D2O and by 1.8 cm-1 for reduced adrenodoxin in D2O. These results suggest that the bridging sulfido groups may be more extensively hydrogen bonded in spinach ferrodoxin and adrenodoxin than in S. platensis ferredoxin, with a further increase in hydrogen-bond strength in the reduced form of adrenodoxin. The similarity of the deuterium effects in the iron-sulfur and blud copper proteins indicates that the deuterium dependence of the blue copper Raman spectra in the 330-460 cm-1 regions is mainly due to hydrogen-bonded cysteinate ligands.

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