Thiol dioxygenases are mononuclear non-heme Fe II -dependent metalloenzymes that initiate the oxidative catabolism of thiol-containing substrates to their respective sulfinates. Cysteine dioxygenase (CDO), the best characterized mammalian thiol dioxygenase, contains a three-histidine (3-His) coordination environment rather than the 2-His-1-carboxylate facial triad seen in most mononuclear non-heme Fe II enzymes. A similar 3-His active site is found in the bacterial thiol dioxygenase 3-mercaptopropionate dioxygenase (MDO), which converts 3-mercaptopropionate into 3-sulfinopropionic acid as part of the bacterial sulfur metabolism pathway. In this study, we have investigated the active site geometric and electronic structures of a third non-heme Fe II -dependent thiol dioxygenase, cysteamine dioxygenase (ADO), by using a spectroscopic approach. Although a 3-His facial triad had previously been implicated on the basis of sequence alignment and site-directed mutagenesis studies, little is currently known about the active site environment of ADO. Our magnetic circular dichroism and electron paramagnetic resonance data provide compelling evidence that ADO features a 3-His facial triad, like CDO and MDO. Despite this similar coordination environment, spectroscopic results obtained for ADO incubated with various substrate analogues are distinct from those obtained for the other Fe II -dependent thiol dioxygenases. This finding suggests that the secondary coordination sphere of ADO is distinct from those of CDO and MDO, demonstrating the significant role that secondary-sphere residues play in dictating substrate specificity.

We report the crystal structure of the mammalian non-heme iron enzyme cysteamine dioxygenase (ADO) at 1.9 Å resolution, which shows an Fe and three-histidine (3-His) active site situated at the end of a wide substrate access channel. The open approach to the active site is consistent with the recent discovery that ADO catalyzes not only the conversion of cysteamine to hypotaurine but also the oxidation of N-terminal cysteine (Nt-Cys) peptides to their corresponding sulfinic acids as part of the eukaryotic N-degron pathway. Whole-protein models of ADO in complex with either cysteamine or an Nt-Cys peptide, generated using molecular dynamics and quantum mechanics/molecular mechanics calculations, suggest occlusion of access to the active site by peptide substrate binding. This finding highlights the importance of a small tunnel that leads from the opposite face of the enzyme into the active site, providing a path through which co-substrate O2 could access the Fe center. Intriguingly, the entrance to this tunnel is guarded by two Cys residues that may form a disulfide bond to regulate O2 delivery in response to changes in the intracellular redox potential. Notably, the Cys and tyrosine residues shown to be capable of forming a cross-link in human ADO reside ∼7 Å from the iron center. As such, cross-link formation may not be structurally or functionally significant in ADO.

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This article describes a graduate student-led effort to develop a climate survey to assess, advocate for, and improve the well-being and mental health of graduate students and postdocs in the Department of Chemistry at the University of Wisconsin−Madison. Graduate students have an increased incidence of depression relative to the general population, and given the transient nature of the student population, understanding and addressing mental health concerns can be challenging. The goal of this article is to illustrate how students, with the support of departmental faculty, staff, and existing on-campus mental health resources, can take the lead to investigate and assess issues related to the challenging graduate school environment. We describe the student-led development and implementation of and the subsequent follow-up to a department-wide survey aimed at destigmatizing the subject of mental health and fostering a more supportive community. This article serves as a framework to assist other interested and motivated graduate students who, with the support of local faculty, wish to develop and initiate a similar process in their own departments. We demonstrate that student-led actions can effectively tackle department-level problems and encourage other interested students to initiate a similar effort.

substrate analogue selenocysteine (Sec)-bound forms of CDO in the Fe(II) and Fe(III) states were investigated spectroscopically. Despite the identical binding modes of Cys and Sec, CDO was found to be unable to oxidize Sec.