Abstract:
:The combination of UV/visible/near-IR variable-temperature magnetic circular 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 transcriptional 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 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that 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 nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain 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 VTMCD 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 other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Dhawan IK,Shelver D,Thorsteinsson MV,Roberts GP,Johnson MKdoi
10.1021/bi991303csubject
Has Abstractpub_date
1999-09-28 00:00:00pages
12805-13issue
39eissn
0006-2960issn
1520-4995pii
bi991303cjournal_volume
38pub_type
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