title: Cysteine dioxygenase structures from pH4 to 9: consistent cys-persulfenate formation at intermediate pH and a Cys-bound enzyme at higher pH.
abstract: Mammalian cysteine dioxygenase (CDO) is a mononuclear non-heme iron protein that catalyzes the conversion of cysteine (Cys) to cysteine sulfinic acid by an unclarified mechanism. One structural study revealed that a Cys-persulfenate (or Cys-persulfenic acid) formed in the active site, but quantum mechanical calculations have been used to support arguments that it is not an energetically feasible reaction intermediate. Here, we report a series of high-resolution structures of CDO soaked with Cys at pH values from 4 to 9. Cys binding is minimal at pH≤5 and persulfenate formation is consistently seen at pH values between 5.5 and 7. Also, a structure determined using laboratory-based X-ray diffraction shows that the persulfenate, with an apparent average O-O separation distance of ~1.8Å, is not an artifact of synchrotron radiation. At pH≥8, the active-site iron shifts from 4- to 5-coordinate, and Cys soaks reveal a complex with Cys, but no dioxygen, bound. This 'Cys-only' complex differs in detail from a previously published 'Cys-only' complex, which we reevaluate and conclude is not reliable. The high-resolution structures presented here do not resolve the CDO mechanism but do imply that an iron-bound persulfenate (or persulfenic acid) is energetically accessible in the CDO active site, and that CDO active-site chemistry in the crystals is influenced by protonation/deprotonation events with effective pKa values near ~5.5 and ~7.5 that influence Cys binding and oxygen binding/reactivity, respectively. Furthermore, this work provides reliable ligand-bound models for guiding future mechanistic considerations.
authors.affiliation: Department of Biochemistry and Biophysics, 2011 Ag and Life Sciences Building, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Camden M
authors.initials: CM
authors.lastname: Driggers
authors.forename: Richard B
authors.initials: RB
authors.lastname: Cooley
authors.forename: Banumathi
authors.initials: B
authors.lastname: Sankaran
authors.forename: Lawrence L
authors.initials: LL
authors.lastname: Hirschberger
authors.forename: Martha H
authors.initials: MH
authors.lastname: Stipanuk
authors.forename: P Andrew
authors.initials: PA
authors.lastname: Karplus
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*; D050558:Cysteine Dioxygenase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: D007501:Iron; D050558:Cysteine Dioxygenase; D003545:Cysteine; C013461:cysteine sulfinic acid
keywords: AML; CDO; LT; PDB; Protein Data Bank; RT; artificial mother liquor; cysteine dioxygenase; high-spin ferrous iron; iron–sulfur; low-temperature; metalloenzyme; room temperature; sulfur metabolism; thiol oxidation