Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy

The combination of UV/visible/near-IR variable-temperature magnetic circula r dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcrip tional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 7 00 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in ox idized wild-type and H77Y CooA. In contrast, the major component in the oxi dized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidin e or histidine/amine axial ligation on the basis of the energy of the porph yrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-1)) and the anisotropy o f the EPR signal, g = 3.23, similar to 2.06, similar to 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate tha t it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrog en ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 a xial ligand switch on reduction of CooA comes from VTMCD studies of the red uced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dom inated by bands characteristic of bis-histidine low-spin Fe(II) hemes, wher eas the reduced H77Y variant is predominantly high-spin with MCD characteri stics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD s tudies show that the CO-bound reduced forms of wild-type, H77Y, and C75S co ntain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMC D spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA, The spectra are almost identical to each o ther and closely correspond to those of the low-temperature photoproducts o f well characterized CO-bound ferrous hemes with His/CO axial ligation.