Probing the Heme Axial Ligation in the CO-Sensing CooA Protein with Magnetic Circular Dichroism Spectroscopy
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- 作者：Ish K. Dhawan ; Daniel Shelver ; Marc V. Thorsteinsson ; Gary P. Roberts ; and Michael K. Johnson
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：September 28, 1999
- 年：1999
- 卷：38
- 期：39
- 页码：12805 - 12813
- 全文大小：112K
- 年卷期：v.38,no.39(September 28, 1999)
- ISSN：1520-4995
文摘
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 theheme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidationtranscriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(
ages/gifchars/pi.gif" BORDER=0 >)-to-Fe(III) charge-transfer band (8930 cm-1) and the presence of cysteinate S-to-Fe(III) charge-transferbands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidizedwild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown tobe a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energyof the porphyrin(ages/gifchars/pi.gif" BORDER=0 >)-to-Fe(III) charge-transfer band (6240 cm-1) and the anisotropy of the EPR signal, g= 3.23, ~2.06, ~1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicatethat it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of ahitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence fora Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reducedproteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristicof bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spinwith MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studiesshow that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemesand that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence thatCO binding to the heme group in reduced CooA occurs with displacement of His77 comes from theVTMCD 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 thelow-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.