Light‐induced oxidation of iron atoms in a photosensitive nitrile hydratase

Abstract

The photoactivation process of a photosensitive nitrilr hydratasc (NHase) from IUtorlococcus sp. N-771 has been invcstigatcd by "Fe M&.sbauer spectroscopy and magnetic susceptibility measurements in order toclarify the behavior of iron atoms in theennyme. MBssbauer spectn of inactive NHase gave two symmetric-doublet components indicating the presence of two iron species, while that of the active NHase gave a single symmetric doublet indicating the presence of a single iron species. Magnetic susceptibility measurcmcnts of the inactive and active HNase both showed small effective magnetic moments. These results led us to conclude that one of the two iron atoms incorporated in the NHase is oxidized during photoactivation, namely from a low spin ferrous 10 a low spin ferric stale. This is the first observation of an intramolecular photooxidation phenomena involving iron in a single protein molecule.

Photosensitive nitrile hydratase intrinsically possesses nitric oxide bound to the non‐heme iron center: evidence by Fourier transform infrared spectroscopy

“…N-771 also possess bacterial NOS, and NO in NHase is produced by this NOS. to Fe(III)Fe(III) upon photoactivation [3]. Since the frequencies of the NO stretching bands are affected by this redox change, as shown in the FTIR difference spectrum (Fig.…”

“…For "N substitution, R. sp. N-771 cells were cultured with growth medium described in [3] except that ("NH&SO, (Shako Co., '<N cnrichment -99%) and Celtone-N (Martek Corp.. "N enrichment -98%) were used as nitrogen sources.…”

“…R312, because the amino acid sequences are identical among the three [2,6,7]. Although various spectroscopies, including EXAFS [8], EPR [9], ENDOR [IO], resonance Raman [8], and Miissbauer [3], have been applied to the NHase from these bacteria, the detailed structure of the di-iron center has not been clarified yet. Also, the mechanism of photoactivation is not well understood except that oxidation of the ferrous iron to the ferric iron takes place within 50 ns upon light irradiation [3,11].…”

“…Although various spectroscopies, including EXAFS [8], EPR [9], ENDOR [IO], resonance Raman [8], and Miissbauer [3], have been applied to the NHase from these bacteria, the detailed structure of the di-iron center has not been clarified yet. Also, the mechanism of photoactivation is not well understood except that oxidation of the ferrous iron to the ferric iron takes place within 50 ns upon light irradiation [3,11]. In this study, by means of light-induced Fourier transform infrared (FTIR) difference spectroscopy (for a recent review of this technique, see [12]), we have found that NHase from R. sp.…”

“…NHase from Rhodococcu.v sp. N-771, which consists of two subunits, a (M, = 22,787) and B (M, = 23,428) [2], possesses two non-heme iron atoms as cofactors [3], and shows a unique manner of photoactivation, i.e. the enzyme is activated by light irradiation [4,5].…”