The core extrusion technique has recently been developed as a chemical method of identification of the type and number (nd, Fe2S2; nt, Fe4S4) of Fe-S sites in proteins. The method is based on removal of Fen Sn core structure (n = 2, 4) with excess thiol in a medium capable of unfolding protein structure, a reaction followed in usual practice by spectrophotometric assay of the extrusion products [Fe2S2(SR)4]2- and/or [Fe2S2(SR)4]2- in the visible region. To circumvent possible interference by protein visible chromophores such as flavins, a Fourier transform (FT) l9F NMR method has been developed using p-CF3C6H4SH (RFSH) as the extrusion reagent. The corresponding extrusion products [Fe2S2(SRF)4]2− and [Fe4S4−(SRF)4]2− exhibit contact-shifted 19F resonances which are separated by ~3 ppm and are well resolved from the RFSH resonance. Control experiments with small ferredoxin proteins have demonstrated that in 4:1 v/v hexamethylphosphoramide/H2O solutions RpSH effects quantitative removal of FenSn cores within the estimated accuracy of the spectrophotometric (±10%) and NMR (±15%) methods. Quantitation in the latter method is achieved by integration of signal intensities after completion of the extrusion reaction and after addition of standard solutions of extrusion products. Application of the method to milk xanthine oxidase (8Fe, 2Mo, 2FAD per Α2 subunit structure) yielded nd = 1.9-2.1 /FAD, showing that all Fe in this enzyme is organized into 4 Fe2S2 centers. Spectrophotometric extrusion of deflavoxanthine oxidase afforded nd = 4.3 based on total Fe, in good agreement with the results of the NMR method. Extrusion reactions of FeMo proteins (23-30 Fe, 1.8-2.0 Mo per Α2β2 subunit structure) of the nitrogenases from Clostridium pasteurianum and Azotobacter vinelandii yielded nt = 1.2-1.4 for the reduced forms and nt = 1.2-1.6, nd = 0.8-1.5 for the thionin-oxidized forms. When normalized to 32Fe/Α2β2 subunit structure, nt = 1.4-2.0 for the reduced forms and nt - 1.4-2.0, nd = 1.0-1.6 for the oxidized forms. Results for the clostridial protein indicated the presence of at least two Fe4S4 sites in both oxidation levels and one Fe2S2 site in the oxidized form. Removal of all Fe not included in the FeMo cofactor was ca. 40-60% complete. The lack of a higher extent of extrusion of noncofactor Fe is attributed to insufficient unfolding of FeMo protein structure by the 4:1 v/v hexamethylphosphoramide/H2O medium. © 1979, American Chemical Society. All rights reserved.
