Improved resolution of 3-mercaptopropionate dioxygenase active site provided by ENDOR spectroscopy offers insight into catalytic mechanism.

3-mercaptopropionate (3MPA) dioxygenase (MDO) is a mononuclear nonheme iron enzyme that catalyzes the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid. MDO is a member of the cysteine dioxygenase family of small molecule thiol dioxygenases and thus shares a conserved sequence of active site residues (Serine-155, Histidine-157, and Tyrosine-159), collectively referred to as the SHY-motif. It has been demonstrated that these amino acids directly interact with the mononuclear Fe-site, influencing steady-state catalysis, catalytic efficiency, O2-binding, and substrate coordination. However, the underlying mechanism by which this is accomplished is poorly understood. Here, pulsed electron paramagnetic resonance spectroscopy [1H Mims electron nuclear double resonance spectroscopy] is applied to validate density functional theory computational models for the MDO Fe-site simultaneously coordinated by substrate and nitric oxide (NO), (3MPA/NO)-MDO. The enhanced resolution provided by electron nuclear double resonance spectroscopy allows for direct observation of Fe-bound substrate conformations and H-bond donation from Tyr159 to the Fe-bound NO ligand. Further inclusion of SHY-motif residues within the validated model reveals a distinct channel restricting movement of the Fe-bound NO-ligand. It has been argued that the iron-nitrosyl emulates the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. While the merit of this assumption remains unconfirmed, the model reported here offers a framework to evaluate oxygen binding at the substrate-bound Fe-site and possible reaction mechanisms. It also underscores the significance of hydrogen bonding interactions within the enzymatic active site.

Cyanide replaces substrate in obligate-ordered addition of nitric oxide to the non-heme mononuclear iron AvMDO active site.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for L-cysteine.

Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster.

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Low-Spin Cyanide Complexes of 3-Mercaptopropionic Acid Dioxygenase (MDO) Reveal the Impact of Outer-Sphere SHY-Motif Residues.

3-Mercaptopropionic acid (3MPA) dioxygenase (MDO) is a non-heme Fe(II)/O2-dependent oxygenase that catalyzes the oxidation of thiol-substrates to yield the corresponding sulfinic acid. Hydrogen-bonding interactions between the Fe-site and a conserved set of three outer-sphere residues (Ser-His-Tyr) play an important catalytic role in the mechanism of this enzyme. Collectively referred to as the SHY-motif, the functional role of these residues remains poorly understood. Here, catalytically inactive Fe(III)-MDO precomplexed with 3MPA was titrated with cyanide to yield a low-spin (S = 1/2) (3MPA/CN)-bound ternary complex (referred to as 1C). UV-visible and electron paramagnetic resonance (EPR) spectroscopy were used to monitor the binding of 3MPA and cyanide. Comparisons of results obtained from SHY-motif variants (H157N and Y159F) were performed to investigate specific H-bonding interactions. For the wild-type enzyme, the binding of 3MPA- and cyanide to the enzymatic Fe-site is selective and results in a homogeneous ternary complex. However, this selectivity is lost for the Y159F variant, suggesting that H-bonding interactions contributed from Tyr159 gate ligand coordination at the Fe-site. Significantly, the g-values for the low-spin ferric site are diagnostic of the directionality of Tyr159 H-bond donation. Computational models coupled with CASSCF/NEVPT2-calculated g-values were used to verify that a major shift in the central g-value (g2) displayed between wild-type and SHY variants could be attributed to the loss of Tyr159 H-bond donation to the Fe-bound cyanide. Applied to native cosubstrate, this H-bond donation provides a means to stabilize Fe-bound dioxygen and potentially explains the attenuated (∼15-fold) rate of catalytic turnover previously reported for MDO SHY-motif variants.

Structure of 3-mercaptopropionic acid dioxygenase with a substrate analog reveals bidentate substrate binding at the iron center.

Thiol dioxygenases are a subset of nonheme iron oxygenases that catalyze the formation of sulfinic acids from sulfhydryl-containing substrates and dioxygen. Among this class, cysteine dioxygenases (CDOs) and 3-mercaptopropionic acid dioxygenases (3MDOs) are the best characterized, and the mode of substrate binding for CDOs is well understood. However, the manner in which 3-mercaptopropionic acid (3MPA) coordinates to the nonheme iron site in 3MDO remains a matter of debate. A model for bidentate 3MPA coordination at the 3MDO Fe-site has been proposed on the basis of computational docking, whereas steady-state kinetics and EPR spectroscopic measurements suggest a thiolate-only coordination of the substrate. To address this gap in knowledge, we determined the structure of Azobacter vinelandii 3MDO (Av3MDO) in complex with the substrate analog and competitive inhibitor, 3-hydroxypropionic acid (3HPA). The structure together with DFT computational modeling demonstrates that 3HPA and 3MPA associate with iron as chelate complexes with the substrate-carboxylate group forming an additional interaction with Arg168 and the thiol bound at the same position as in CDO. A chloride ligand was bound to iron in the coordination site assigned as the O2-binding site. Supporting HYSCORE spectroscopic experiments were performed on the (3MPA/NO)-bound Av3MDO iron nitrosyl (S = 3/2) site. In combination with spectroscopic simulations and optimized DFT models, this work provides an experimentally verified model of the Av3MDO enzyme-substrate complex, effectively resolving a debate in the literature regarding the preferred substrate-binding denticity. These results elegantly explain the observed 3MDO substrate specificity, but leave unanswered questions regarding the mechanism of substrate-gated reactivity with dioxygen.

Corneal donations in South Africa: A 15-year review.

BACKGROUND: Corneal pathology is one of the leading causes of preventable blindness in South Africa (SA). A corneal transplant can restore or significantly improve vision in most cases. However, in SA there is a gross shortage of corneal tissue available to ophthalmologists. Little has been published describing the magnitude of the problem. OBJECTIVES: To describe trends in the number of corneal donors per year in SA, the number of corneal transplants performed each year, the origin of donors, the allocation of corneas to the public or private sector, and the demographics of donors. METHODS: This was a retrospective review of all corneal donations to SA eye banks over the 15-year period 1 January 2002 - 31 December 2016. RESULTS: There was a progressive year-on-year decline in corneal donors over the study period, from 565 per year in 2002 to 89 in 2016. As a direct result, there has been an 85.5% decrease in the number of corneal transplants performed per year using locally donated corneas, from 1 049 in 2002 to 152 in 2016. Of the donors, 48.8% originated from mortuaries, 39.0% from private hospitals and 12.2% from government hospitals; donors from mortuaries showed the most significant decline over the 15-year period, decreasing by 94.8%. Of donated corneas, 79.3% were allocated to the private sector and 21.7% to the public sector. Males comprised 69.1% of donors, while 77.2% were white, 14.0% coloured, 6.3% black and 2.5% Indian/Asian. Donor age demonstrated a bimodal peak at 25 and 55 years. CONCLUSIONS: The number of corneal donations in SA has declined markedly, causing the burden of corneal disease requiring transplantation to rise steadily. Population groups with a low donor rate may have cultural and other objections to corneal donation, which should be a major focus of future research and initiatives aimed at reversing the current trends.