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Cysteine dioxygenase structures from pH 4 to 9: consistent Cys-persulfenate formation at intermediate pH and a Cys-bound enzyme at higher pH

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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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  • Driggers, C.M., Cooley, R.B., Sankaran, B., Hirschberger, L.L, Stipanuk, M.H., & Karplus, P.A. (2013). Cysteine dioxygenase structures from pH 4 to 9: Consistent cys-persulfenate formation at intermediate pH and a cys-bound enzyme at higher pH. Journal of Molecular Biology, 425(17), 3121-3136. doi:10.1016/j.jmb.2013.05.028
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  • 425
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  • 17
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  • Journal of Molecular Biology - Elsevier
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  • This project was supported in part by Grant DK-056649 to MHS from the National Institute of Diabetes and Digestive and Kidney Diseases. It was also aided by the Collaborative Crystallography Project of the Berkeley Center for Structural Biology supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. Synchrotron data were collected at the Advanced Light Source and the NSLS, respectively supported by contracts DE-AC02-98CH10886 and DE-AC02-05CH11231 from the Office of Basic Energy Sciences of the U.S. Department of Energy
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