Interspin distances in spin-labeled metmyoglobin variants determined by saturation recovery EPR.
Saturation recovery (SR) electron paramagnetic resonance was used to determine the distance between iron and nitroxyl for spin-labeled metmyoglobin variants in low-spin and high-spin states of the Fe(III). The interspin distances were measured by analyzing the effect of the heme iron on the spin-lattice relaxation rates of the nitroxyl spin label using the modified Bloembergen equation for low-spin species, and an analogue of the Bloembergen equation for high-spin species. Insight simulations of the spin-labeled protein structures also were used to determine the interspin distances. The distances obtained by SR for high-spin and low-spin complexes with 15-20 A interspin distances, for low-spin CN(-) and high-spin formate adducts at distances up to about 30 A, and results from Insight calculations were in good agreement. For variants with 25-30 A interspin distances, the distances obtained by SR for the fluoride adducts were shorter than observed for the CN(-) or formate adducts or predicted by Insight simulations. Of the heme axial ligands examined (CN(-), imidazole, F(-), and formate), CN(-) is the best choice for determination of iron-nitroxyl distances in the range of 15-30 A.
Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208-2436, USA.
Y
Y
Zhou
B E
BE
Bowler
K
K
Lynch
S S
SS
Eaton
G R
GR
Eaton
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008786:Metmyoglobin / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels; D014907:Whales
D008024:Ligands; D009211:Myoglobin; D011994:Recombinant Proteins; D013113:Spin Labels; D008786:Metmyoglobin; D006418:Heme; D007501:Iron