Electron-electron distances in spin-labeled low-spin metmyoglobin variants by relaxation enhancement.
Thirteen single-cysteine variants of myoglobin were prepared by overexpression of apoprotein, spin labeling, and reconstitution with hemin. This procedure resulted in a protein with fewer hemichrome impurities than was obtained by an overexpression of holo-protein followed by spin labeling. Coordination of cyanide to the met heme formed low-spin complexes. Iron-nitroxyl interspin distances in the range of 17-30 A were determined by saturation recovery measurements of the enhancement of the nitroxyl spin lattice relaxation rates between approximately 30-140 K, and by spin-echo measurements of the enhancement of spin-spin relaxation rates at 10-30 K. Interspin distances were also calculated, using the molecular modeling program Insight II (Accelrys, San Diego, CA). For most variants, distances determined from the temperature dependence of spin-echo intensities at a pulse spacing of 200 ns agree with distances measured by saturation recovery and calculated with Insight II within about an angstrom, which is within experimental uncertainties. Measurements of interspin distances via spin-spin relaxation enhancement have the advantages that maximum effects are observed for slower metal relaxation rates than are required for spin-lattice relaxation enhancement, and the impact diminishes as r(-3) instead of r(-6), as with spin-lattice relaxation enhancement, which permits measurements at longer distances.
Ulyanov
Dmitriy
D
Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208-2436, USA.
Bowler
Bruce E
BE
Eaton
Gareth R
GR
Eaton
Sandra S
SS
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D008786:Metmyoglobin / Q000737:chemistry* / Q000235:genetics*; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D013113:Spin Labels; D013696:Temperature; D013997:Time Factors
D050505:Mutant Proteins; D013113:Spin Labels; D008786:Metmyoglobin
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