Identification of Two Important Heme Site Residues (Cysteine 75 and Histidine 77) in CooA, the CO-Sensing Transcription Factor of Rhodospirillum rubrum
The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a...
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Published in | Biochemistry (Easton) Vol. 38; no. 9; pp. 2669 - 2678 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
02.03.1999
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Subjects | |
Online Access | Get full text |
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Summary: | The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding. |
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Bibliography: | istex:701948D10958777CFA71C14B553E649A0B13D084 Supported by Grants GM 53228 (G.P.R.) and HL 54762 (J.N.B). from the National Institutes of Health and by fellowship support from the Alfred P. Sloan Foundation (J.N.B.) and NIH Biophysics Training Grant T32 GM 08293 (M.F.R.). ark:/67375/TPS-6X8GM46F-Q ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi982658j |