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

Abstract

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 34S shows that they arise from cysteine coordinated to the ferric 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 15N 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 34S 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 2H isotope shifts seen after exchange of the protein into 2H2O 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 et 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.