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KarplusPAndrewBiochemistryBiophysicsCysteineDioxygenaseStructures(SupplementaryInformation).pdf

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  • Mammalian cysteine dioxygenase (CDO) is a mononuclear non-heme iron protein that catalyzes 2 the conversion of cysteine (Cys) to cysteine sulfinic acid (CSA) by an unclarified mechanism. 3 One structural study revealed a Cys-persulfenate (or Cys-persulfenic acid) formed in the active 4 site, but quantum mechanical calculations have been used to support arguments that it is not an 5 energetically feasible reaction intermediate. Here, we report a series of high-resolution structures 6 of CDO soaked with Cys at pH values from 4 to 9. Cys binding is minimal at pH≤5 and 7 persulfenate formation is consistently seen at pH values between 5.5 and 7. Also, a structure 8 determined using laboratory-based X-ray diffraction shows that the persulfenate, with an 9 apparent average O-O separation distance of ~1.8 Å is not an artifact of synchrotron radiation. At 10 pH≥8, the active site iron shifts from 4- to 5-coordinate, and Cys soaks reveal a complex with 11 Cys, but no dioxygen, bound. This ‘Cys-only’ complex differs in detail from a previously 12 published ‘Cys-only’ complex which we reevaluate and conclude is not reliable. The high-13 resolution structures presented here do not resolve the CDO mechanism, but do imply that an 14 iron-bound persulfenate (or persulfenic acid) is energetically accessible in the CDO active site, 15 and that CDO active site chemistry in the crystals is influenced by protonation/deprotonation 16 events with effective pKa values near ~5.5 and ~7.5 that influence Cys binding and oxygen 17 binding/reactivity, respectively. Furthermore, this work provides reliable ligand-bound models 18 for guiding future mechanistic considerations.
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