Functional Complementation Studies Reveal Different Interaction Partners of <i>Escherichia coli</i> IscS and Human NFS1

The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron–sulfur (Fe–S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human l-cysteine desulfurase NFS1 and the <i>Escherichia coli</i> homologue IscS share a level of amino acid sequence identity of ∼60%. While <i>E. coli</i> IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe–S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm⁵s²U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an <i>E. coli iscS</i> deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and <i>E. coli</i> IscS share conserved binding sites for proteins involved in Fe–S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present

The Role of SufS Is Restricted to Fe–S Cluster Biosynthesis in <i>Escherichia coli</i>

In <i>Escherichia coli</i>, two different systems that are important for the coordinate formation of Fe–S clusters have been identified, namely, the ISC and SUF systems. The ISC system is the housekeeping Fe–S machinery, which provides Fe–S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for several sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS leads to an overall decrease in Fe–S cluster enzyme and molybdoenzyme activity in addition to a decrease in the number of Fe–S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe–S-dependent. Complementation of the ISC deficient strains with the <i>suf</i> operon restored the activity of Fe–S-containing proteins, including the MoaA protein, which is involved in the conversion of 5′GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share a high degree of similarity, we show that the function of their respective l-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot play the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe–S cluster assembly in the cell

Low-Potential Amperometric Enzyme Biosensor for Xanthine and Hypoxanthine

The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and also xanthine to uric acid by an oxidative hydroxylation mechanism. The present electrochemical biosensor was optimized in terms of applied potential and pH. The electrocatalytic oxidation response showed a linear dependence on the xanthine concentration ranging from 1.0 × 10^–5 to 1.8 × 10^–3 M with a correlation coefficient of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM (signal-to-noise ratio = 3) using controlled potential amperometry

Catalytic Electrochemistry of Xanthine Dehydrogenase

We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from <i>Rhodobacter capsulatus</i> at a thiol-modified Au electrode. The 2-electron acceptor <i>N</i>-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD⁺. XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed

Pyranopterin Dithiolene Distortions Relevant to Electron Transfer in Xanthine Oxidase/Dehydrogenase

The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo^IV-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase. These Mo^IV-product complexes display an intense metal-to-ligand charge-transfer (MLCT) band in the near-infrared region of the spectrum. Optical pumping into this MLCT band yields resonance Raman spectra of the Mo site that are devoid of contributions from the highly absorbing FAD and 2Fe2S clusters in the protein. The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes. This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity

The N‑Terminus of Iron–Sulfur Cluster Assembly Factor ISD11 Is Crucial for Subcellular Targeting and Interaction with l‑Cysteine Desulfurase NFS1

Assembly of iron–sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by l-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondrial targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved “LYR” motif is essential for the role of ISD11 in forming a stable and active complex with NFS1

Functional Studies on <i>Oligotropha carboxidovorans</i> Molybdenum–Copper CO Dehydrogenase Produced in <i>Escherichia coli</i>

The Mo/Cu-dependent CO dehydrogenase (CODH) from <i>Oligotropha carboxidovorans</i> is an enzyme that is able to catalyze both the oxidation of CO to CO₂ and the oxidation of H₂ to protons and electrons. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of <i>O. carboxidovorans</i> CODH in <i>Escherichia coli</i>. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from <i>O. carboxidovorans</i>. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H₂ oxidation of Mo/Cu-dependent CODHs in the future

Vibrational Probes of Molybdenum Cofactor–Protein Interactions in Xanthine Dehydrogenase

The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen-bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco–protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme–product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the −NH₂ terminus of the PDT, and between Q197 and the terminal oxo of the MoO group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters <i>k</i>_red and <i>k</i>_red/<i>K</i>_D, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes

Complementary Surface-Enhanced Resonance Raman Spectroscopic Biodetection of Mixed Protein Solutions by Chitosan- and Silica-Coated Plasmon-Tuned Silver Nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome <i>b</i>₅, whereas functionalization with SiO₂ amplifies only the spectra of the cationic protein cytochrome <i>c</i>. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy

Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X‑ray Absorption Spectroscopy

Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium <i>Rhodobacter capsulatus</i>, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5–10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo­(VI) ion with each one MPT, MoO, Mo–O^–, and MoS ligand, and a weak Mo–O­(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (p<i>K</i> ∼ 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (p<i>K</i> ∼ 8.8) is an inhibitor at alkaline pH. A similar acidic p<i>K</i> for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo–OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation