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In mammals, the non-heme iron enzyme cysteine dioxygenase (CDO) helps regulate Cys levels through converting Cys to Cys sulfinic acid. Its activity is in part modulated by the formation of a Cys93–Tyr157 crosslink that increases its catalytic efficiency over 10-fold. Here, 21 high-resolution mammalian CDO structures are used to gain insight into how the Cys–Tyr crosslink promotes activity and how select competitive inhibitors bind. Crystal structures of crosslink-deficient C93A and Y157F variants reveal similar ~ 1.0-Å shifts in the side chain of residue 157, and both variant structures have a new chloride ion coordinating the active site iron. Cys binding is also different from wild-type CDO, and no Cys-persulfenate forms in the C93A or Y157F active sites at pH 6.2 or 8.0. We conclude that the crosslink enhances activity by positioning the Tyr157 hydroxyl to enable proper Cys binding, proper oxygen binding, and optimal chemistry. In addition, structures are presented for homocysteine (Hcy), D-Cys, thiosulfate, and azide bound as competitive inhibitors. The observed binding modes of Hcy and D-Cys clarify why they are not substrates, and the binding of azide shows that in contrast to what has been proposed, it does not bind in these crystals as a superoxide mimic. [ABSTRACT FROM AUTHOR]

In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15-30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue ('Arg-type' enzymes) and some having a Gln substituted for this Arg ('Gln-type' enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis 'Arg-type' enzyme that has cysteine dioxygenase activity ( BsCDO), and a Ralstonia eutropha 'Gln-type' CDO homolog of uncharacterized activity ( ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among 'Gln-type' CDO enzymes, we conclude that the 'Gln-type' CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases. [ABSTRACT FROM AUTHOR]

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 pK a 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. [Copyright &y& Elsevier]