A Look at Electron Pulse Paramagnetic Resonance Spectroscopy: An Interview with Molly Lockart and Brad Pierce

Molly Lockart is a professor in the Department of Chemistry and Biochemistry at Samford University in Homewood, Alabama, and Brad Pierce is a professor in the Department of Chemistry and Biochemistry at the University of Alabama. Recently, Lockart and Pierce published a study that investigated the mechanism of 3-mercaptopropionate dioxygenase (MDO), an enzyme that plays a role in the oxidation of thiol-bearing compounds. MDO contains a specific sequence of active site residues known as the SHY-motif, which interacts with the iron site in the enzyme (1). However, the precise mechanism by which this interaction occurs is not well understood. Using pulsed electron paramagnetic resonance spectroscopy, the researchers validated computational models of the MDO iron site coordinated with both substrate and nitric oxide (NO), referred to as (3MPA/NO)-MDO (1).

Lockart and Pierce sat down with Spectroscopy to talk about their group’s work in this space, and how their work helps us to understand more about how interactions within the enzymatic active site can influence the mechanism of sulfur-oxidation and how enzyme dysfunction can result in disease states.

Professors Molly Lockart (left) of Samford University and Brad S. Pierce (right) of the University of Alabama. Photo Credit: © Molly Lockart and Brad S. Pierce

Would you explain what pulsed electron paramagnetic resonance (EPR) spectroscopy is and its main applications for readers who may be unfamiliar with this analytical technique?

Pulsed EPR is a spectroscopic technique that probes a paramagnetic compound using short, high-powered microwave pulses. Varying the microwave pulse sequence and pulse length allows us to selectively detect magnetic interactions from nearby nuclei or other unpaired spins, which reveals nuanced details about molecular structure and function.

Can you explain the role of 3-mercaptopropionate (3MPA) dioxygenase (MDO) in catalyzing the oxidation of thiol-bearing substrates, and what is its significance in biological systems?

MDO is a member of the small-molecule thiol dioxygenase (smTDO) family. These enzymes utilize a mononuclear ion within their active site to catalyze the O₂-dependent oxidation of organic thiol-substrates to yield the corresponding sulfinic acid.

Although MDO is a bacterial enzyme, the active site architecture is similar to mammalian thiol dioxygenases such as cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO). Among humans, changes in the activity, or cellular expression of CDO and ADO, have been correlated with disease states such as cancer, neurodegenerative disorders, rheumatoid arthritis, and other metabolic disorders.

The overarching goal of this work was to elucidate key substrate interactions within the enzymatic active site that influence the mechanism of sulfur-oxidation. This is a crucial first step in understanding how dysfunction of these enzymes can result in disease states.

Your research study mentions the SHY-motif (Serine155, Histidine-157, and Tyrosine-159) as crucial for MDO activity. Could you elaborate on how these residues interact with the mononuclear Fe-site and influence catalysis?

The “SHY-motif” refers to three amino acids in the MDO active site (Serine-155, Histidine-157, and Tyrosine-159), which form a hydrogen-bonding network terminating at the mononuclear iron site. These residues are highly conserved among smTDOs across all phylogenic domains of life.

Multiple research groups have demonstrated that disruption of the SHY-motif hydrogen-bonding network dramatically decreases enzymatic activity. However, the exact function of these residues in facilitating catalytic turnover is poorly understood. A significant hurdle in understanding the function of the SHY-motif is the absence of crystallographic data for MDO in complex with its native substrate (3-mercaptopropionate, 3MPA). In the absence of this structural information, the nature of interactions between the SHY-motif and the substrate-bound Fe-site were unknown.

In this work, we used a combination of spectroscopy and computational methods to provide a structure of the substrate-bound active site. Significantly, we directly observed hydrogen bond donation from the terminal Tyrosine-159 of the SHY-motif to the axial position at the Fe-site. This suggests that the SHY-motif directly influences the mechanism of oxygen addition to the substrate-thiol.

How does pulsed electron paramagnetic resonance spectroscopy contribute to our understanding of the MDO Fe-site, particularly in the context of substrate and nitric oxide (NO) coordination?

The NO-bound Fe-site is similar in size and geometry to the proposed iron(III)-superoxide transient intermediate involved in substrate-oxidation. However, the iron-nitrosyl site is stable and can be spectroscopically characterized by pulsed EPR.

In this work, pulsed electron nuclear double resonance (ENDOR) spectroscopy was applied to structurally interrogate the MDO active site simultaneously bound by nitric oxide and 3-mercaptopropionate, termed (3MPA/NO)-MDO. Using this pulsed EPR method, the strength of interaction between the paramagnetic iron-nitrosyl and selected magnetic nuclei (¹H) within the MDO active site was measured. Because the magnitude of this interaction scales inversely with distance, ENDOR can be used to measure specific distances within the enzymatic active site.

The study discusses the observation of Fe-bound substrate conformations and hydrogen bond donation from Tyr159 to Fe-bound NO (1). Can you explain the significance of these observations in the context of MDO catalytic activity?

The added resolution of ENDOR spectroscopy allowed us to validate our computational models of substrate positioning and the orientation of the axial NO ligand. As structure determines function, proper modeling of the substrate-bound active site is essential for evaluating possible reaction mechanisms. By focusing on the positions of protons on 3MPA and the Tyr159-OH, we built on our previous understanding of the orientation of 3MPA relative to the Fe-site and the importance of the SHY-motif in substrate binding and reactivity.

It is also worth mentioning that the experiments presented to discriminate between structural conformers were performed to provide an analytic benchmark to demonstrate the structural resolution of the ENDOR method. As noted in the manuscript (1), previous work using another pulsed EPR method referred to as hyperfine sublevel correlation spectroscopy (HYSCORE) was unable to differentiate the substrate conformers.

The inclusion of SHY-motif residues in the validated model reveals a distinct channel restricting the movement of the Fe-bound NO-ligand. What implications does this restriction have on the catalytic cycle of MDO?

We found that the position of the NO ligand is constrained in a channel between His142 and Tyr159, where it is in an optimal position to maintain H-bond contact with Tyr159. In addition, we demonstrated that the rotation of the Fe-bound NO ligand is energetically uphill. This positioning challenges a sequential oxygenation mechanism and instead supports a direct attack of the proximal O-atom in the superoxide intermediate, though further work is necessary to fully understand the overall oxygenation mechanism.

The study suggests that the iron-nitrosyl complex may emulate the structure of potential Fe(III)-superoxide intermediates within the MDO catalytic cycle. How does this assumption contribute to our understanding of MDO's mechanism of action?

No intermediates in the MDO catalytic cycle have been previously reported. However, an iron(III)-superoxide intermediate has been reported for CDO is structurally similar to MDO (2,3).

Because this intermediate is short-lived, structural characterization of this intermediate is not feasible. However, the iron-nitrosyl is very stable, allowing for spectroscopic characterization. Furthermore, the similar size and geometry of the iron-nitrosyl make it an ideal candidate to model oxygen binding at the substrate-bound active site. From this investigation, we were able to carefully evaluate the likelihood of mechanistic pathways proposed for this enzyme. Specifically, our structural model suggests that the proposed sequential addition of dioxygen is unlikely given the energetically unfavorable rotation necessary to place the distal O-atom in the superoxide intermediate close enough to the S-atom on 3MPA for the sequential addition to occur.

In what ways does the validated model reported in the study provide a framework for evaluating oxygen binding at the substrate-bound Fe-site and potential reaction mechanisms?

The spectroscopically validated model gives us a glimpse into the catalytic cycle and adds new details about the assembly of the O₂-reactive Fe site that were not previously understood. The added resolution of 3MPA positioning and Tyr-159 H-bond donation to the Fe-site builds on our previous understanding of the catalytic mechanism and informs future work to better understand the overall mechanism of smTDOs.

The study underscores the significance of hydrogen bonding interactions within the enzymatic active site. Could you discuss how these interactions contribute to MDO's catalytic efficiency and substrate specificity?

Our combined spectroscopic and computational approach verified that H-bond donation to the axial ligand was necessary for catalysis and that the orientation of the axial ligand is in part determined by the Tyr159 H-bond. In addition, this work built on our previous conclusion that H-bond donation to the Fe-site modulated ligand binding and could play a role in the obligate-ordered addition of substrate prior to O₂.

Are there any practical applications or implications of the findings reported in this study for fields such as biotechnology, the pharmaceutical industry, or medicine?

A critical first objective in the rational design of any therapeutic agent targeting small molecule TDOs is the determination of the structure for the oxygen:substrate:enzyme ternary complex. This objective is followed closely by the identification of catalytically essential interactions within the active site. This work advances both objectives.

The development of new therapeutic agents to treat imbalances in sulfur metabolism relies on a thorough understanding of the enzymatic mechanisms involved in sulfur oxidation. This work provided structural models to evaluate potential reaction mechanisms for smTDOs. It also provided the first molecular-level insight into the function of the highly conserved SHY-motif.

This interview has been lightly edited for clarity.

References

(1) Pierce, B. S.; Schmittou, A. N.; York, N. J. Improved Resolution of 3-Mercaptopropionate Dioxygenase Active Site Provided by ENDOR Spectroscopy Offers Insight into Catalytic Mechanism. J. Bio. Chem. 2024, 300 (4), 105777. DOI: 10.1016/j.jbc.2024.105777

(2) Crawford J.A.; Li, W.; Pierce, B.S. Single turnover of substrate-bound ferric cysteine dioxygenase with superoxide anion: enzymatic reactivation, product formation, and a transient intermediate. Biochemistry 2011, 50 (47), 10241.

(3) Tchesnokov, E.P.; Faponle, A.S.; Davies, C.G.; et al. An Iron-Oxygen Intermediate Formed During the Catalytic Cycle of Cysteine Dioxygenase. Chem. Commun. 2016, 52 (57), 8814. DOI: 10.1039/C6CC03904A