Spectroscopic and Functional Characterization of the Nitric Oxide Adducts of the Fe(III) and Fe(II) States of the CO-Sensing Transcription Factor, CooA, from Carboxydothermus Hydrogenoformans

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CooA is a carbon monoxide (CO) sensing transcription factor that is found in several bacteria and regulates CO metabolism. CO binding to CooA’s heme groups initiates a conformation change that activates protein DNA binding. This study investigates the reaction of Fe(III) Carboxydothermus hydrogenoformansCooA (Ch CooA) with nitric oxide (NO). Previously, Clark and coworkers reported Fe(II) Ch CooA bound NO to form a 6-coordinate (6-C) Fe(II)-NO adduct that was active for DNA binding in vivo and in vitro. This is in contrast to the best studied CooA homolog from R. rubrum (Rr) that was exquisitely specific for CO and formed an inactive 5-coordinate (5-C) Fe(II)-NO adduct. In the current study, when Fe(III) Ch CooA was incubated with NO, a final 5-C Fe(II)-NO species resulted. This species had an electronic absorbance spectrum similar to Rr CooA; however, unlike Rr CooA, the 5-C Fe(II)-NO Ch CooA adduct retained some DNA-binding activity. Results from mutagenesis studies suggest that S-nitrosylation of a Cys residue located in the heme pocket may account for the unexpected reactivity differences of Fe(III) and Fe(II) Ch CooA with NO. Based on these data, it is proposed the ability of Ch CooA (and failure of Rr CooA) to be activated by NO is due to the binding of NO to the correct (i.e. distal) heme face. The authors wish to thank the Valparaiso University College of Arts and Sciences for financial support.