STRUCTURE OF THE PHOTOREACTIVE IRON CENTER OF THE NITRILE HYDRATASE FROM RHODOCOCCUS SP. N-771 - EVIDENCE OF A NOVEL POSTTRANSLATIONAL MODIFICATION IN THE CYSTEINE LIGAND

Nitrile hydratase (NHase) from Rhodococcus sp, N-771 is a photoreactiv e enzyme that is inactivated by nitrosylation of the non-heme iron cen ter and activated by photodissociation of nitric oxide (NO), To obtain structural information on the iron center, we isolated peptide comple xes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the pep tide fragment, -Thr-Ala-Trp-Pro-Ile-Pro-Pro-Thr-Trp-Tyr-Lys(128). The peptide contained 0.79 mol of iron/mol of molecule as well as endogeno us NO. Subsequently, by digesting the peptide with thermolysin, carbox ypeptidase Y, and leucine aminopeptidase hi, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 a mino acid residues from alpha lle(107) to alpha Trp(117), Furthermore, by using mass spectrometry, protein sequence, and amino acid composit ion analyses, we have shown that the 112th Cys residue of the cu subun it is post-translationally oxidized to a cysteine-sulfinic acid (Cys-S O2H) in the NHase, These results indicate that the NHase from Rhodococ cus sp, N-771 has a novel non-heme iron enzyme containing a cysteine-s ulfinic acid in the iron center, Possible ligand residues of the iron center are discussed.