title: pH dependence of cyanide binding to the ferric heme domain of the direct oxygen sensor from Escherichia coli and the effect of alkaline denaturation.
abstract: The spectrum of the ferric heme domain of the direct oxygen sensor protein from Escherichia coli ( EcDosH) has been measured between pH 3.0 and 12.6. EcDosH undergoes acid denaturation with an apparent p K a of 4.24 +/- 0.05 and a Hill coefficient of 3.1 +/- 0.6 and reversible alkaline denaturation with a p K a of 9.86 +/- 0.04 and a Hill coefficient of 1.1 +/- 0.1. Cyanide binding to EcDosH has been investigated between pH 4 and 11. The EcDosH-cyanide complex is most stable at pH 9 with a K D of 0.29 +/- 0.06 microM. The kinetics of cyanide binding are monophasic between pH 4 and 8. At pH >or=8.5, the reaction is biphasic with the fast phase dependent upon the cyanide concentration and the slow phase independent of cyanide. The slow phase is attributed to conversion of denatured EcDosH to the native state, with a pH-independent rate of 0.052 +/- 0.006 s (-1). The apparent association rate constant for cyanide binding to EcDosH increases from 3.6 +/- 0.1 M (-1) s (-1) at pH 4 to 520 +/- 20 M (-1) s (-1) at pH 11. The dissociation rate constant averages (8.6 +/- 1.3) x 10 (-5) s (-1) between pH 5 and 9, increasing to (1.4 +/- 0.1) x 10 (-3) s (-1) at pH 4 and (2.5 +/- 0.1) x 10 (-3) s (-1) at pH 12.2. The mechanism of cyanide binding is consistent with preferential binding of the cyanide anion to native EcDosH. The reactions of imidazole and H 2O 2 with ferric EcDosH were also investigated and show little reactivity.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115, USA.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Bidwai
authors.forename: Esther Y
authors.initials: EY
authors.lastname: Ok
authors.forename: James E
authors.initials: JE
authors.lastname: Erman
mesh_terms: D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003486:Cyanides / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation
chemical_list: D002352:Carrier Proteins; D003486:Cyanides; D029968:Escherichia coli Proteins; D006420:Hemeproteins; C406529:dosH protein, E coli; D006418:Heme
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