Abstract
The techniques of EPR and electron nuclear double resonance (ENDOR) were used to probe structure and electronic distribution at the nitric oxide (NO)-ligated heme alpha 3 in the nitrosylferrocytochrome alpha 3 moiety of fully reduced cytochrome c oxidase. Hyperfine and quadrupole couplings to NO (in both 15NO and 14NO forms), to histidine nitrogens, and to protons near the heme site were obtained. Parallel studies were also performed on NO-ligated myoglobin and model NO-heme-imidazole systems. The major findings and interpretations on nitrosylferrocytochrome alpha 3 were: 1) compared to other NO-heme-imidazole systems, the nitrosylferrocytochrome alpha3 gave better resolution of EPR and ENDOR signals; 2) at the maximal g value (gx = 2.09), particularly well resolved NO nitrogen hyperfine and quadrupole couplings and mesoproton hyperfine couplings were seen. These hyperfine and quadrupole couplings gave information on the electronic distribution on the NO, on the orientation of the g tensor with respect to the heme, and possibly on the orientation of the FeNO plane; 3) a combination of experimental EPR-ENDOR results and EPR spectral simulations evidenced a rotation of the NO hyperfine tensor with respect to the electronic g tensor; this implied a bent Fe-NO bond; 4) ENDOR showed a unique proton not seen in the other NO heme systems studied. The magnitude of this proton's hyperfine coupling was consistent with this proton being part of a nearby protein side chain that perturbs an axial ligand like NO or O2.
Highlights
The techniques of EPR and electron nuclear double resonance (ENDOR) were used to probe structure and electronic distribution at the nitric oxide (NO)-ligated heme a3 in the nitrosylferrocytochrome a3 moiety of fully reduced cytochrome c oxidase
2.09), well resolved NO nitrogen hyperfine and quadrupole couplings and mesoproton hyperfine couplings were seen. These hyperfine and quadrupole couplings gave information on the electronic distribution on the NO, on the orientation of the g tensor with respect to the heme, and possibly on the orientation of the FeNO plane; 3) a combination of experimental results and EPR spectral simulations evidenced a rotation of the NO hyperfine tensor with respect to the electronic g tensor; this implied a bent Fe-NO bond; 4) ENDOR showed a unique proton not seen in the other NO heme systems studied
U z 2 cn a LARGE COUPLING- 29.86 spectrum from g = 2.09 to g = 1.97, we found that therewere ENDOR resonances (Table I, B and C) ofNO which stayed essentially at the same minimal frequencies found at g, ( i e . near 21 MHz for ' W O and near 15 MHz for I4NO).Quadrupole couplings wereresolved only at g, for the 14N0
Summary
There was no EPR evidence for oxidized cytochrome a with its low spin ferric heme a and Cu. signals. A similar procedure was Instrumentation-EPR spectra were obtained with a Bruker ER- used for the preparation of NO-Mb derivatives, except that for 420 spectrometer equipped for low power dispersion operation and reduction of Mb a 1.5 molar excess (relative to heme) of sodium equipped with a home-built low temperature microwave head and dithionite (Hardman& Holden, Ltd., Manchester, England) was used. Hemewas distortion, standard absorption mode first derivative spectra (dx"/ anaerobically dissolved in DMF (99.5%deuterated, Merck) and a 60-. EPR spectra were digitized resultant six-coordinate NO-heme-imidazole complex (as readily and double integrated, and the results were compared to a 1 mM noted from its EPR spectrum) was tipped into the EPR tuhe and. EPR frequencies and magnetic field quickly frozen in liquid N2
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
