Structural measurements in hemoprotiens: use of spin-labeled protoheme as a probe of heme environment.

With the aid of two kinds of spin-labeled protohemins, the nature of the heme-protein interaction of various hemoproteins was investigated. Di- and mono-spin-labeled protohemins were prepared from protohemin and 2,2,5,5-tetramethyl-3-aminopyrrolidine-1-oxyl. The spin-labeled hemins were recombined with apoproteins of hemoglobin (Hb), myoglobin (Mb), cytochrome c peroxidase (EC 1.11.1.5) and horseradish peroxidase (EC 1.11.1.7). Electron paramagnetic resonance spectra of the di- and mono-spin-labeled hemoglobin in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees C exhibited moderate immobilization of the labels, while that of cytochrome c peroxidase showed stronger immobilization. Di-spin-labeled horseradish peroxidase showed an EPR spectrum of a simple broad line with peak-to-peak line width of 35 G. This broadening is due to spin-spin interaction between the two labels attached at the 6- and 7-positions of the porphyrin ring. Ligand binding to the spin-labeled hemoproteins altered the EPR line shapes and amplitudes. The former is attributed to the changes in the mobility of the labels and the latter to the magnetic dipolar interaction between the heme iron and free radical. From the strength of this interaction the distance between the iron and the nitroxide radical may be calculated. In the hemoproteins examined, the distances are: Hb 12.5 A, Mb 12.0 A, cytochrome peroxidase approximately 14 A, and horseradish peroxidase 9.0 A.