Resonance Raman spectroscopy of nitrile hydratase, a novel iron - Sulfur enzyme

Resonance Raman spectra of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase, a novel non-heme iron enzyme, have a large number of peaks in the 300-500 cm(-1) region; observation of shifts in these peaks after labeling with S-34 shows that they arise from cysteine coordinated to the fen-ic ion In the protein. The rich Raman spectra result from coupling of the Fe-S stretch with cysteine side chain deformation modes; the observation of N-15 isotope shifts in most of these peaks suggests participation of N-donor metal ligands and peptide backbone amide nitrogens in these modes as well. The aggregate S-34 isotope shift is too large to result from a single cysteine ligand, consistent with the analysis of EXAFS data that shows two or three S-donor ligands [Scarrow et al. (1996) Biochemistry 35, 10078-10088]. Widespread H-2 isotope shifts seen after exchange of the protein into (H2O)-H-2 suggest the presence of hydrogen bonds to the coordinated cysteine sulfurs. Comparison of the resonance Raman spectra of nitrile hydratase prepared at pH 7.3 and 9.0 shows a shift of intensity into the higher-energy peaks in the spectra of the latter sample, This is interpreted as resulting from an increase in Fe-S bond strength at the higher pH and is supported by observation of a small decrease in Fe-S bond length in the EXAFS analysis [Scarrow el al. (1996) Biochemistry 35, 10078-10088]. Such a decrease in Fe-S bond length is also consistent with pH dependent changes in EPR spectra and could reflect the loss of one or more hydrogen bonds to sulfur ligands.