Resonance Raman studies in some carboni anhydrase-aromatic sulfonamide complexes.

Resonance Raman spectra of 4-sulfonamido -4'-dimethylaminoazobenzene, 4-sulfonamido-4'-hydroxyazobenzene, and 4-sulfonamideo-4' aminoazobenzene bound to various isoenzymes of carbonic anhydrase were obtained by exciting into the sulfonamide absorption bands in the 400-500-nm region. In this way it was possible to obtain vibrational spectra of the sulfonamides in the active site unmasked by contributions from the vibrational modes of the protein and H2O solvent. Direct evidence was obtained for the presence of -SO2NH- in the complex, and it was possible to eliminate hydrophobic bonding and twisting in the Ph--N==N--Ph bonds as sources of the observed spectral changes. No detectable differences were found in the spectra of 4-sulfonamido-4'-dimethylaminoazobenzene bound to human carbonic anhydrase B and C or between these and the spectra of the sulfonamide bound to bovine carbonic anhydrase or Co(II) human carbonic anhydrase B. A new band appears in the spectra of the bound sulfonamides, and this is interpreted in terms of a change in geometry about the sulfonamido sulfur atom. A possible explanation for this change in geometry is that the bound sulfonamide group closely mimics the transition state of the reactants in the reversible hydration of CO2. Sulfonamide binding at pH 12.0 was not detected in the resonance Raman spectrum. Sulfonamide replacement of CN- in the binding site occurred over a period of minutes and could be monitored in the resonance Raman spectra.