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.

Understanding the diverse reactivities of vitamin B12 and its derivatives, collectively called cobalamins, requires detailed knowledge of their geometric and electronic structures. Electronic absorption (Abs) and resonance Raman (rR) spectroscopies have proven invaluable in this area, particularly when used in concert with computational techniques such as density functional theory (DFT). There remain, however, lingering uncertainties in the computational description of electronic excited states of cobalamins, particularly surrounding the vibronic coupling that impacts the Abs bandshapes and gives rise to rR enhancement of vibrational modes. Past computational analyses of the vibrational spectra of cobalamins have either neglected rR enhancement or calculated rR enhancement for only a small number of modes. In the present study, we used the recently developed ORCA_ASA computational tool in conjunction with the popular B3LYP and BP86 functionals to predict Abs bandshapes and rR spectra for vitamin B12. The ORCA_ASA/B3LYP-computed Abs envelope in the visible spectral region and rR spectra of vitamin B12 agree remarkably well with our experimental data, while BP86 fails to reproduce both. This finding represents a significant advance in our understanding of how these two commonly used density functionals differently model the electronic properties of cobalamins. Guided by the computed frequencies for the Co–C stretching and Co–C–N bending modes, we identified, for the first time, isotope-sensitive features in our rR spectra of 12CNCbl and 13CNCbl that can be assigned to these modes. A normal coordinate analysis of the experimentally determined Co–C stretching and Co–C–N bending frequencies indicates that the Co–C force constant for vitamin B12 is 2.67 mdyn/Å, considerably larger than the Co–C force constants reported for alkylcobalamins.

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