Resonance Raman evidence that photodissociation of nitric oxide from the non-heme iron center activates nitrile hydratase from Rhodococcus sp. N-771.
Nitrile hydratase (NHase) from Rhodococcus sp. N-771, which contains a non-heme iron center in the catalytic site, has been known to be activated by light illumination. Recently, endogenous nitric oxide (NO) was found in this enzyme by FTIR spectroscopy [Noguchi et al. (1995) FEBS Lett. 358, 9-12]. In order to directly detect the bonding between NO and the iron atom and the reaction of NO upon photoactivation, resonance Raman spectra of the NHase were measured with 413 nm excitation at 85 K. Two prominent bands at 592 and 570 cm-1 were observed in the inactive from, and both of them were completely lost upon photoactivation. Upon subsequent introduction of 15NO, the active NHase was converted to the inactive form again and the above two bands were restored with downshifts by 10 and 12 cm-1, respectively. Also, the excitation profiles of these bands in the 350-500 nm region mostly followed the absorption spectrum arising from the iron center. From these isotopic shifts and the excitation profiles, the two Raman bands were assigned to the Fe-NO stretching and bending vibrations that are probably coupled with each other. The results provided solid evidence that NO is bound to the non-heme iron in the inactive NHase and its photodissociation activates the enzyme.
Noguchi
T
T
Photosynthesis Research Laboratory, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Hoshino
M
M
Tsujimura
M
M
Odaka
M
M
Inoue
Y
Y
Endo
I
I
D001665:Binding Sites; D004789:Enzyme Activation / Q000528:radiation effects; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D007501:Iron / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry; D010777:Photochemistry; D012240:Rhodococcus / Q000201:enzymology*; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman
D009569:Nitric Oxide; D007501:Iron; D006836:Hydro-Lyases; C048831:nitrile hydratase