Preparation and characterization of MnIIMnIII complexes with relevance to class Ib ribonucleotide reductases.

The Mn2 complex [MnII2(TPDP)(O2CPh)2](BPh4) (1, TPDP = 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol, Ph =phenyl) was prepared and subsequently characterized via single-crystal X-ray diffraction, X-ray absorption, electronic absorption, and infrared spectroscopies, and mass spectrometry. 1 was prepared in order to explore its properties as a structural and functional mimic of class Ib ribonucleotide reductases (RNRs). 1 reacted with superoxide anion (O2•-) to generate a peroxido-MnIIMnIII complex, 2. The electronic absorption and electron paramagnetic resonance (EPR) spectra of 2 were similar to previously published peroxido-MnIIMnIII species. Furthermore, X-ray near edge absorption structure (XANES) studies indicated the conversion of a MnII2 core in 1 to a MnIIMnIII state in 2. Treatment of 2 with para-toluenesulfonic acid (p-TsOH) resulted in the conversion to a new MnIIMnIII species, 3, rather than causing O-O bond scission, as previously encountered. 3 was characterized using electronic absorption, EPR, and X-ray absorption spectroscopies. Unlike other reported peroxido-MnIIMnIII species, 3 was capable of oxidative O-H activation, mirroring the generation of tyrosyl radical in class Ib RNRs, however without accessing the MnIIIMnIV state.

Protein engineering a PhotoRNR chimera based on a unifying evolutionary apparatus among the natural classes of ribonucleotide reductases.

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating ß subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y356[ß], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y356• along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any ß metallocofactor highlights the adaptability of the 10-stranded αß barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs.

Evaluation of 1,10-phenanthroline-based hydroxamate derivative as dual histone deacetylases/ribonucleotide reductase inhibitor with antitumor activities.

BACKGROUND: Aberrant expression of histone deacetylases (HDACs) and ribonucleotide reductase (RR) enzymes are commonly observed in various cancers. Researchers are focusing on these enzymes in cancer studies with the aim of developing effective chemotherapeutic drugs for cancer treatment. Targeting both HDAC and RR simultaneously with a dual HDAC/RR inhibitor has exhibited enhanced effectiveness compared to monotherapy in cancer treatment, making it a promising strategy. OBJECTIVES: The objective of the study is to synthesize and assess the anti-cancer properties of a 1,10-phenanthroline-based hydroxamate derivative, characterizing it as a novel dual HDAC/RR inhibitor. METHODS: The N1-hydroxy-N8-(1,10-phenanthrolin-5-yl)octanediamide (PA), a 1,10-phenanthroline-based hydroxamate derivative, was synthesized and structurally characterized. The compound was subjected to in vitro assessments of its anti-cancer, HDAC, and RR inhibitory activities. In silico docking and molecular dynamics simulations were further studied to explore its interactions with HDACs and RRM2. RESULTS: The structurally confirmed PA exhibited antiproliferative activity in SiHa cells with an IC50 of 16.43 µM. It displayed potent inhibitory activity against HDAC and RR with IC50 values of 10.80 µM and 9.34 µM, respectively. Co-inhibition of HDAC and RR resulted in apoptosis-induced cell death in SiHa cells, mediated by the accumulation of reactive oxygen species (ROS). In silico docking studies demonstrated that PA can effectively bind to the active sites of HDAC isoforms and RRM2. Furthermore, PA demonstrated a more favorable interaction with HDAC7, displaying a docking score of -9.633 kcal/mol, as compared to the standard HDAC inhibitor suberoylanilide hydroxamic acid (SAHA), which exhibited a docking score of -8.244 kcal/mol against HDAC7. CONCLUSION: The present study emphasizes the prospect of designing a potential 1,10-phenanthroline hydroxamic acid derivative as a novel dual HDAC and RR-inhibiting anti-cancer molecule.

Structure of a ribonucleotide reductase R2 protein radical.

Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O-O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.

Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR requires two homodimeric subunits, α and ß. The active form is an asymmetric αα'ßß' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C439•), and the ß subunit houses the diferric-tyrosyl radical (Y122•) that is essential for C439• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y122•[ß] ↔ W48?[ß] ↔ Y356[ß] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]. In a recent cryo-EM structure, Y356[ß] was revealed for the first time and it, along with Y731[α], spans the asymmetric α/ß interface. An E52[ß] residue, which is essential for Y356 oxidation, allows access to the interface and resides at the head of a polar region comprising R331[α], E326[α], and E326[α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y356• was photochemically generated using a photosensitizer covalently attached adjacent to Y356[ß]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E52[ß], R331[α], E326[α], and E326[α'] network plays the essential role of shuttling protons associated with Y356 oxidation from the interface to bulk solvent.

Site-Differentiated MnIIFeII Complex Reproducing the Selective Assembly of Biological Heterobimetallic Mn/Fe Cofactors.

Class Ic ribonucleotide reductases (RNRIc) and R2-like ligand-binding oxidases (R2lox) are known to contain heterobimetallic MnIIFeII cofactors. How these enzymes assemble MnIIFeII cofactors has been a long-standing puzzle due to the weaker binding affinity of MnII versus FeII. In addition, the heterobimetallic selectivity of RNRIc and R2lox has yet to be reproduced with coordination complexes, leading to the hypothesis that RNRIc and R2lox overcome the thermodynamic preference for coordination of FeII over MnII with their carefully constructed three-dimensional protein structures. Herein, we report the selective formation of a heterobimetallic MnIIFeII complex accomplished in the absence of a protein scaffold. Treatment of the ligand Py4DMcT (L) with equimolar amounts of FeII and MnII along with two equivalents of acetate (OAc) affords [LMnIIFeII (OAc)2(OTf)]+ (MnIIFeII) in 80% yield, while the diiron complex [LFeIIFeII(OAc)2(OTf)]+ (FeIIFeII) is produced in only 8% yield. The formation of MnIIFeII is favored regardless of the order of addition of FeII and MnII sources. X-ray diffraction (XRD) of single crystals of MnIIFeII reveals an unsymmetrically coordinated carboxylate ligand─a primary coordination sphere feature shared by both RNRIc and R2lox that differentiates the two metal binding sites. Anomalous XRD studies confirm that MnIIFeII exhibits the same site selectivity as R2lox and RNRIc, with the FeII (d6) center preferentially occupying the distorted octahedral site. We conclude that the successful assembly of MnIIFeII originates from (1) Fe-deficient conditions, (2) site differentiation, and (3) the inability of ligand L to house a dimanganese complex.

The Chlamydia trachomatis p-aminobenzoate synthase CADD is a manganese-dependent oxygenase that uses its own amino acid residues as substrates.

CADD (chlamydia protein associating with death domains) is a p-aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope-labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen. Further, mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, providing strong evidence that this residue is sacrificed for pAB synthesis. Additionally, Lys152 was deaminated and oxidized to aminoadipic acid, supporting its proposed role as a sacrificial amino group donor.

Starting a new chapter on class Ia ribonucleotide reductases.

Ribonucleotide reductases (RNRs) use radical-based chemistry to convert ribonucleotides into deoxyribonucleotides, an essential step in DNA biosynthesis and repair. There are multiple RNR classes, the best studied of which is the class Ia RNR that is found in Escherichia coli, eukaryotes including humans, and many pathogenic and nonpathogenic prokaryotes. This review covers recent advances in our understanding of class Ia RNRs, including a recent reporting of a structure of the active state of the E. coli enzyme and the impacts that the structure has had on spurring research into the mechanism of long-range radical transfer. Additionally, the review considers other recent structural and biochemical research on class Ia RNRs and the potential of that work for the development of anticancer and antibiotic therapeutics.

Ferritin-Like Proteins: A Conserved Core for a Myriad of Enzyme Complexes.

Ferritin-like proteins share a common fold, a four α-helix bundle core, often coordinating a pair of metal ions. Although conserved, the ferritin fold permits a diverse set of reactions, and is central in a multitude of macromolecular enzyme complexes. Here, we emphasize this diversity through three members of the ferritin-like superfamily: the soluble methane monooxygenase, the class I ribonucleotide reductase and the aldehyde deformylating oxygenase. They all rely on dinuclear metal cofactors to catalyze different challenging oxygen-dependent reactions through the formation of multi-protein complexes. Recent studies using cryo-electron microscopy, serial femtosecond crystallography at an X-ray free electron laser source, or single-crystal X-ray diffraction, have reported the structures of the active protein complexes, and revealed unprecedented insights into the molecular mechanisms of these three enzymes.

Still no Rest for the Reductases: Ribonucleotide Reductase (RNR) Structure and Function: An Update.

Herein we present a multidisciplinary discussion of ribonucleotide reductase (RNR), the essential enzyme uniquely responsible for conversion of ribonucleotides to deoxyribonucleotides. This chapter primarily presents an overview of this multifaceted and complex enzyme, covering RNR's role in enzymology, biochemistry, medicinal chemistry, and cell biology. It further focuses on RNR from mammals, whose interesting and often conflicting roles in health and disease are coming more into focus. We present pitfalls that we think have not always been dealt with by researchers in each area and further seek to unite some of the field-specific observations surrounding this enzyme. Our work is thus not intended to cover any one topic in extreme detail, but rather give what we consider to be the necessary broad grounding to understand this critical enzyme holistically. Although this is an approach we have advocated in many different areas of scientific research, there is arguably no other single enzyme that embodies the need for such broad study than RNR. Thus, we submit that RNR itself is a paradigm of interdisciplinary research that is of interest from the perspective of the generalist and the specialist alike. We hope that the discussions herein will thus be helpful to not only those wanting to tackle RNR-specific problems, but also those working on similar interdisciplinary projects centering around other enzymes.

Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b-NrdI complex monitored by serial femtosecond crystallography.

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b-NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b-NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins.

Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2-like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radical reactivity of R2c from the C-H activation reactions of R2lox remain unknown. In this work, multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy and ligand hyperfine techniques in conjunction with broken-symmetry density functional theory calculations are used to characterize the molecular and electronic structures of two EPR-active intermediates trapped during aerobic assembly of the R2lox Mn/Fe cofactor. A MnIII(µ-O)(µ-OH)FeIII species is identified as the first EPR-active species and represents a common state between the two classes of redox-active Mn/Fe proteins. The species downstream from the MnIII(µ-O)(µ-OH)FeIII state exhibits unique EPR properties, including unprecedented spectral breadth and isotope-dependent g-tensors, which are attributed to a weakly coupled, hydrogen-bonded MnIII(µ-OH)FeIII species. This final intermediate precedes formation of the MnIII/FeIII resting state and is suggested to be relevant to understanding the endogenous reactivity of R2lox.

HUG Domain Is Responsible for Active Dimer Stabilization in an NrdJd Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/ß Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2ß2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and ß, to initiate, using its metallo-cofactor in ß2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(ß) and Y731(α) across the α/ß interface. Using 2,3,5-F3Y122-ß2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-ß2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Kinetic model for reversible radical transfer in ribonucleotide reductase.

The enzyme ribonucleotide reductase (RNR), which catalyzes the reduction of ribonucleotides to deoxynucleotides, is vital for DNA synthesis, replication, and repair in all living organisms. Its mechanism requires long-range radical translocation over ∼32 Šthrough two protein subunits and the intervening aqueous interface. Herein, a kinetic model is designed to describe reversible radical transfer in Escherichia coli RNR. This model is based on experimentally studied photoRNR systems that allow the photochemical injection of a radical at a specific tyrosine residue, Y356, using a photosensitizer. The radical then transfers across the interface to another tyrosine residue, Y731, and continues until it reaches a cysteine residue, C439, which is primed for catalysis. This kinetic model includes radical injection, an off-pathway sink, radical transfer between pairs of residues along the pathway, and the conformational flipping motion of Y731 at the interface. Most of the input rate constants for this kinetic model are obtained from previous experimental measurements and quantum mechanical/molecular mechanical free-energy simulations. Ranges for the rate constants corresponding to radical transfer across the interface are determined by fitting to the experimentally measured Y356 radical decay times in photoRNR systems. This kinetic model illuminates the time evolution of radical transport along the tyrosine and cysteine residues following radical injection. Further analysis identifies the individual rate constants that may be tuned to alter the timescale and probability of the injected radical reaching C439. The insights gained from this kinetic model are relevant to biochemical understanding and protein-engineering efforts with potential pharmacological implications.

Role of Water in Proton-Coupled Electron Transfer between Tyrosine and Cysteine in Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides and is critical for DNA synthesis and repair in all organisms. Its mechanism requires radical transfer along a ∼32 Špathway through a series of proton-coupled electron transfer (PCET) steps. Previous simulations suggested that a glutamate residue (E623) mediates the PCET reaction between two stacked tyrosine residues (Y730 and Y731) through a proton relay mechanism. This work focuses on the adjacent PCET reaction between Y730 and a cysteine residue (C439). Quantum mechanical/molecular mechanical free energy simulations illustrate that when Y730 and Y731 are stacked, E623 stabilizes the radical on C439 through hydrogen bonding with the Y730 hydroxyl group. When Y731 is flipped away from Y730, a water molecule stabilizes the radical on C439 through hydrogen bonding with Y730 and lowers the free energy barrier for radical transfer from Y730 to C439 through electrostatic interactions with the transferring hydrogen but does not directly accept the proton. These simulations indicate that the conformational motions and electrostatic interactions of the tyrosines, cysteine, glutamate, and water strongly impact the thermodynamics and kinetics of these two coupled PCET reactions. Such insights are important for protein engineering efforts aimed at altering radical transfer in RNR.

TAT-RHIM: a more complex issue than expected.

Murine cytomegalovirus protein M45 contains a RIP homotypic interaction motif (RHIM) that is sufficient to confer protection of infected cells against necroptotic cell death. Mechanistically, the N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils, and interacts with the endogenous RHIM domains of receptor-interacting serine/threonine protein kinases (RIPK) 1, RIPK3, Z-DNA-binding protein 1, and Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-ß. Remarkably, all four aforementioned mammalian proteins harbouring such a RHIM domain are key components of inflammatory signalling and regulated cell death (RCD) processes. Immunogenic cell death by regulated necrosis causes extensive tissue damage in a wide range of diseases, including ischaemia reperfusion injury, myocardial infarction, sepsis, stroke, and solid organ transplantation. To harness the cell death suppression properties of M45 protein in a therapeutically usable manner, we developed a synthetic peptide encompassing only the RHIM domain of M45. To trigger delivery of RHIM into target cells, we fused the transactivator protein transduction domain of human immunodeficiency virus 1 to the N-terminus of the peptide. The fused peptide could efficiently penetrate eukaryotic cells, but unexpectedly it eradicated or destroyed all tested cancer cell lines and primary cells irrespective of species without further stimulus through a necrosis-like cell death. Typical inhibitors of different forms of RCD cannot impede this process, which appears to involve a direct disruption of biomembranes. Nevertheless, our finding has potential clinical relevance; reliable induction of a necrotic form of cell death distinct from all known forms of RCD may offer a novel therapeutic approach to combat resistant tumour cells.

Effects of chameleon dispense-to-plunge speed on particle concentration, complex formation, and final resolution: A case study using the Neisseria gonorrhoeae ribonucleotide reductase inactive complex.

Ribonucleotide reductase (RNR) is an essential enzyme that converts ribonucleotides to deoxyribonucleotides and is a promising antibiotic target, but few RNRs have been structurally characterized. We present the use of the chameleon, a commercially-available piezoelectric cryogenic electron microscopy plunger, to address complex denaturation in the Neisseria gonorrhoeae class Ia RNR. Here, we characterize the extent of denaturation of the ring-shaped complex following grid preparation using a traditional plunger and using a chameleon with varying dispense-to-plunge times. We also characterize how dispense-to-plunge time influences the amount of protein sample required for grid preparation and preferred orientation of the sample. We demonstrate that the fastest dispense-to-plunge time of 54 ms is sufficient for generation of a data set that produces a high quality structure, and that a traditional plunging technique or slow chameleon dispense-to-plunge times generate data sets limited in resolution by complex denaturation. The 4.3 Å resolution structure of Neisseria gonorrhoeae class Ia RNR in the inactive α4ß4 oligomeric state solved using the chameleon with a fast dispense-to-plunge time yields molecular information regarding similarities and differences to the well studied Escherichia coli class Ia RNR α4ß4 ring.

Structural and Biochemical Investigation of Class I Ribonucleotide Reductase from the Hyperthermophile Aquifex aeolicus.

Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed of two proteins, a large α-subunit (R1) and a smaller ß-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. Aquifex aeolicus is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the A. aeolicus R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive A. aeolicus RNR. While the active oligomeric state and activity regulation of A. aeolicus RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a ß-hairpin hook structure at the dimer interface, which has an interesting π-stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of A. aeolicus in low-oxygen environments.

Ribonucleotide reductase: In-vitro S-glutathionylation of R2 and p53R2 subunits of mammalian class I ribonucleotide reductase protein.

Ribonucleotide reductases (RNR) catalyze the rate-limiting step in DNA synthesis during the S-phase of the cell cycle. Its constant activity in order to maintain dNTP homeostasis is a fascinating area of research and an attractive candidate for cancer research and antiviral drugs. Redox modification such as S-glutathionylation of the R1 subunit of mammalian RNR protein has been presumed to regulate the activity of RNR during catalytic cycles. Herein, we report S-glutathionylation of the R2 subunit. We have also shown Grx1 system can efficiently deglutathionylate the S-glutathionylated R2 subunit. Additionally, our data also showed for the very first time S-glutathionylation of mammalian p53R2 subunit that regulates DNA synthesis outside S-phase during DNA damage and repair. Taken together, these data will open new avenues for future research relating to exact physiological significance, target thiols, and/or overall RNR activity due to S-glutathionylation of R2 and p53R2 subunits and provide valuable insights for effective treatment regimes.