Copper(II) Affects the Biochemical Behavior of Proinsulin C-peptide by Forming Ternary Complexes with Serum Albumin.

Peptide hormones are essential signaling molecules with therapeutic importance. Identifying regulatory factors that drive their activity gives important insight into their mode of action and clinical development. In this work, we demonstrate the combined impact of Cu(II) and the serum protein albumin on the activity of C-peptide, a 31-mer peptide derived from the same prohormone as insulin. C-peptide exhibits beneficial effects, particularly in diabetic patients, but its clinical use has been hampered by a lack of mechanistic understanding. We show that Cu(II) mediates the formation of ternary complexes between albumin and C-peptide and that the resulting species depend on the order of addition. These ternary complexes notably alter peptide activity, showing differences from the peptide or Cu(II)/peptide complexes alone in redox protection as well as in cellular internalization of the peptide. In standard clinical immunoassays for measuring C-peptide levels, the complexes inflate the quantitation of the peptide, suggesting that such adducts may affect biomarker quantitation. Altogether, our work points to the potential relevance of Cu(II)-linked C-peptide/albumin complexes in the peptide's mechanism of action and application as a biomarker.

Characterizing metal-biomolecule interactions by mass spectrometry.

Metal micronutrients are essential for life and exist in a delicate balance to maintain an organism's health. The labile nature of metal-biomolecule interactions clouds the understanding of metal binders and metal-mediated conformational changes that are influential to health and disease. Mass spectrometry (MS)-based methods and technologies have been developed to better understand metal micronutrient dynamics in the intra- and extracellular environment. In this review, we describe the challenges associated with studying labile metals in human biology and highlight MS-based methods for the discovery and study of metal-biomolecule interactions.

Differences in the Second Coordination Sphere Tailor the Substrate Specificity and Reactivity of Thiol Dioxygenases.

In recent years, considerable progress has been made toward elucidating the geometric and electronic structures of thiol dioxygenases (TDOs). TDOs catalyze the conversion of substrates with a sulfhydryl group to their sulfinic acid derivatives via the addition of both oxygen atoms from molecular oxygen. All TDOs discovered to date belong to the family of cupin-type mononuclear nonheme Fe(II)-dependent metalloenzymes. While most members of this enzyme family bind the Fe cofactor by two histidines and one carboxylate side chain (2-His-1-carboxylate) to provide a monoanionic binding motif, TDOs feature a neutral three histidine (3-His) facial triad. In this Account, we present a bioinformatics analysis and multiple sequence alignment that highlight the significance of the secondary coordination sphere in tailoring the substrate specificity and reactivity among the different TDOs. These insights provide the framework within which important structural and functional features of the distinct TDOs are discussed.The best studied TDO is cysteine dioxygenase (CDO), which catalyzes the conversion of cysteine to cysteine sulfinic acid in both eukaryotes and prokaryotes. Crystal structures of resting and substrate-bound mammalian CDOs revealed two surprising structural motifs in the first- and second coordination spheres of the Fe center. The first is the presence of the abovementioned neutral 3-His facial triad that coordinates the Fe ion. The second is the existence of a covalent cross-link between the sulfur of Cys93 and an ortho carbon of Tyr157 (mouse CDO numbering scheme). While the exact role of this cross-link remains incompletely understood, various studies established that it is needed for proper substrate Cys positioning and gating solvent access to the active site. Intriguingly, bacterial CDOs lack the Cys-Tyr cross-link; yet, they are as active as cross-linked eukaryotic CDOs.The other known mammalian TDO is cysteamine dioxygenase (ADO). Initially, it was believed that ADO solely catalyzes the oxidation of cysteamine to hypotaurine. However, it has recently been shown that ADO additionally oxidizes N-terminal cysteine (Nt-Cys) peptides, which indicates that ADO may play a much more significant role in mammalian physiology than was originally anticipated. Though predicted on the basis of sequence alignment, site-directed mutagenesis, and spectroscopic studies, it was not until last year that two crystal structures, one of wild-type mouse ADO (solved by us) and the other of a variant of nickel-substituted human ADO, finally provided direct evidence that this enzyme also features a 3-His facial triad. These structures additionally revealed several features that are unique to ADO, including a putative cosubstrate O2 access tunnel that is lined by two Cys residues. Disulfide formation under conditions of high O2 levels may serve as a gating mechanism to prevent ADO from depleting organisms of Nt-Cys-containing molecules.The combination of kinetic and spectroscopic studies in conjunction with structural characterizations of TDOs has furthered our understanding of enzymatic sulfhydryl substrate regulation. In this article, we take advantage of the fact that the ADO X-ray crystal structures provided the final piece needed to compare and contrast key features of TDOs, an essential family of metalloenzymes found across all kingdoms of life.

The Crystal Structure of Cysteamine Dioxygenase Reveals the Origin of the Large Substrate Scope of This Vital Mammalian Enzyme.

We report the crystal structure of the mammalian non-heme iron enzyme cysteamine dioxygenase (ADO) at 1.9 Šresolution, 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 Šfrom the iron center. As such, cross-link formation may not be structurally or functionally significant in ADO.

Spectroscopic investigation of iron(III) cysteamine dioxygenase in the presence of substrate (analogs): implications for the nature of substrate-bound reaction intermediates.

Thiol dioxygenases (TDOs) are a class of metalloenzymes that oxidize various thiol-containing substrates to their corresponding sulfinic acids. Originally established by X-ray crystallography for cysteine dioxygenase (CDO), all TDOs are believed to contain a 3-histidine facial triad that coordinates the necessary Fe(II) cofactor. However, very little additional information is available for cysteamine dioxygenase (ADO), the only other mammalian TDO besides CDO. Previous spectroscopic characterizations revealed that ADO likely binds substrate cysteamine in a monodentate fashion, while a mass spectrometry study provided evidence that a thioether crosslink can form between Cys206 and Tyr208 (mouse ADO numbering). In the present study, we have used electronic absorption and electron paramagnetic resonance (EPR) spectroscopies to investigate the species formed upon incubation of Fe(III)ADO with sulfhydryl-containing substrates and the superoxide surrogates azide and cyanide. Our data reveal that azide is unable to coordinate to cysteamine-bound Fe(III)ADO, suggesting that the Fe(III) center lacks an open coordination site or azide competes with cysteamine for the same binding site. Alternatively, cyanide binds to either cysteamine- or Cys-bound Fe(III)ADO to yield a low-spin (S = 1/2) EPR signal that is distinct from that observed for cyanide/Cys-bound Fe(III)CDO, revealing differences in the active-site pockets between ADO and CDO. Finally, EPR spectra obtained for cyanide/cysteamine adducts of wild-type Fe(III)ADO and its Tyr208Phe variant are superimposable, implying that either an insignificant fraction of as-isolated wild-type enzyme is crosslinked or that formation of the thioether bond has minimal effects on the electronic structure of the iron cofactor.

Spectroscopic Investigation of Cysteamine Dioxygenase.

Thiol dioxygenases are mononuclear non-heme FeII-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 FeII 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 FeII-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 FeII-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.