Cysteine Ligands Research Articles

Cytochrome P450-The Wonderful Nanomachine Revealed through Dynamic Simulations of the Catalytic Cycle.

This Account addresses the catalytic cycle of the enzyme cytochrome P450 (CYP450) as a prototypical biological machine with automatic features. CYP450 is a nanomachine that uses dioxygen and two reducing and two proton equivalents to oxidize a plethora of molecules (so-called substrates) as a means of supplying bio-organisms with essential molecules (e.g., brain neurotransmitters, sex hormones, etc.) and protecting biosystems against poisoning. An enticing property of CYP450s is that entrance of an oxidizable substrate into the active site initiates a series of events that constitute the catalytic cycle, which functions "automatically" in a regulated sequence of events culminating in the production of the oxidized substrates (e.g., hydroxylated, epoxidized, etc.), oftentimes with remarkable stereo- and regioselectivities. It is timely to demonstrate how theory uses molecular dynamics (MD) simulations and quantum-mechanical/molecular-mechanical (QM/MM) calculations to complement experiments and elucidate the choreography by which the protein regulates the catalytic cycle. CYP450 is a heme enzyme that contains a ferric ion (FeIII) coordinated by a porphyrin ligand, a water molecule, and a cysteinate ligand that is provided by a strategic residue of the encapsulating protein. While many of the individual steps are sufficiently well-understood, we shall provide here an overview of the factors that cause all of the steps to be sequentially coordinated. To this end, we use examples from three different CYP450 enzymes: the bacterial ones CYP450BM3 and CYP450CAM and the mammalian enzyme CYP4503A4. The treatment is limited to the catalytic cycle, as aspects of two-state reactivity were reviewed previously (e.g., Shaik , S. ; et al. Chem. Rev. 2005 , 105 , 2279 ). What are the principles that govern the seeming automatic feature? For example, how do substrate entrance and binding gate the enzyme? How does the reductase attachment to the enzyme affect the next steps? What triggers the attachment of the reductase? How does the electron transfer (ET) that converts FeIII to FeII occur? Is the ET coordinated with the entrance of O2 into the active site? What is the mechanism of the latter step? Since the entrance of the substrate expels the water molecules from the active site, how do water molecules re-enter to form a proton channel, which is necessary for creating the ultimate oxidant Compound I? How do mutations that disrupt the water channel nevertheless create a competent oxidant? By what means does the enzyme produce regio- and stereoselective oxidation products? What triggers the departure of the oxidized product, and how does the exit occur in a manner that generates the resting state ready for the next cycle? This Account shows that the entrance of the substrate triggers all of the ensuing events.

Solution NMR Spectroscopy with Isotope-Labeled Cysteine (13C and 15N) Reveals the Surface Structure of l-Cysteine-Coated Ultrasmall Gold Nanoparticles (1.8 nm).

Ultrasmall gold nanoparticles with a diameter of 1.8 nm were synthesized by reduction of tetrachloroauric acid with sodium borohydride in the presence of l-cysteine, with natural isotope abundance as well as 13C-labeled and 15N-labeled. The particle diameter was determined by high-resolution transmission electron microscopy and differential centrifugal sedimentation. X-ray photoelectron spectroscopy confirmed the presence of metallic gold with only a few percent of oxidized Au(+I) species. The surface structure and the coordination environment of the cysteine ligands on the ultrasmall gold nanoparticles were studied by a variety of homo- and heteronuclear NMR spectroscopic techniques including 1H-13C-heteronuclear single-quantum coherence and 13C-13C-INADEQUATE. Further information on the binding situation (including the absence of residual or detached l-cysteine in the solution) and on the nanoparticle diameter (indicating the well-dispersed state) was obtained by diffusion-ordered spectroscopy (1H-, 13C-, and 1H-13C-DOSY). Three coordination environments of l-cysteine on the gold surface were identified that were ascribed to different crystallographic sites, supported by geometric considerations of the nanoparticle ultrastructure. The particle size data and the NMR-spectroscopic analysis gave a particle composition of about Au174(cysteine)67.

Evaluation of modified hyaluronic acid in terms of rheology, enzymatic degradation and mucoadhesion

PurposeThis study aimed to investigate the properties of modified hyaluronic acid in terms of rheological properties, enzymatic degradation and mucoadhesiveness. MethodsHyaluronic acid (HA) was chemically modified with sulfhydryl ligand cysteine ethyl ester (C) in order to immobilize sulfhydryl groups on the polymeric backbone. MTT assay was performed to evaluate the safety of hyaluronic acid-cysteine ethyl ester (HAC). Rheological and enzymatic degradation studies were accomplished by preparing hydrogels of HA and HAC, respectively. HA served as control. Enzymes such as lysozyme, amylase and hyaluronidase were chosen to perform degradation studies. To study mucoadhesiveness, hydrogels of HA and HAC, respectively, were mixed with mucus and evaluated by rheology. ResultsMTT assay indicated no toxicity at all. The rheological assay showed 2.2-fold increase in gelling properties in case of HAC in comparison to HA. Furthermore, it could be shown that HAC was degraded by amylase to a lesser extent of 11.5-fold than HA. After 2 h, HA showed a higher degradation by lysozyme with 67.97% than HAC. Adhesion studies exhibited a 2.17-fold higher mucoadhesion of HAC with mucus compared to HA. ConclusionThese results will open the door for high efficient drug delivery systems based on hydrogels for mucosal application.

Learning from diminutive ligand design

Protein Design One strategy for understanding the origin of life is proposing simple replacements for the complex biomolecules that have developed through billions of years of evolution. Ferredoxins are small proteins that contain simple, cubic clusters of iron and sulfur atoms and act as mobile electron carriers in cells. Kim et al. designed a 12-residue peptide with alternating D and L amino acids that can replicate the placement of cysteine ligands found in many natural ferredoxins. After reconstitution with iron and sulfur, the peptides bound a single iron-sulfur cluster. The resulting minimal, artificial ferredoxin exhibited a redox potential compatible with some biological processes. J. Am. Chem. Soc. 140 , 11210 (2018).

The Role of Mixed Amine/Amide Ligation in Nickel Superoxide Dismutase.

Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redox-active metal cycles between oxidized and reduced forms that differ by one electron to catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. Nickel-dependent SOD (NiSOD) is a unique biological solution for controlling superoxide levels. This enzyme relies on the use of cysteinate ligands to bring the Ni(III/II) redox couple into the range required for catalysis (∼300 mV vs. NHE). The use of cysteine thiolates, which are not found in any other SOD, is a curious choice because of their well-known oxidation by peroxide and dioxygen. The NiSOD active site cysteinate ligands are resistant to oxidation, and prior studies of synthetic and computational models point to the backbone N-donors in the active site (the N-terminal amine and the amide N atom of Cys2) as being involved in stabilizing the cysteines to oxidation. To test the role of the backbone N-donors, we have constructed a variant of NiSOD wherein an alanine residue was added to the N-terminus (Ala0-NiSOD), effectively altering the amine ligand to an amide. X-ray absorption, electronic absorption, and magnetic circular dichroism (MCD) spectroscopic analyses of as-isolated Ala0-NiSOD coupled with density functional theory (DFT) geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and time-dependent DFT computations are consistent with a diamagnetic Ni(II) site with two cysteinate, one His1 amide, and one Cys2 amidate ligands. The variant protein is catalytically inactive, has an altered electronic absorption spectrum associated with the nickel site, and is sensitive to oxidation. Mass spectrometric analysis of the protein exposed to air shows the presence of a mixture of oxidation products, the principal ones being a disulfide, a bis-sulfenate, and a bis-sulfinate derived from the active site cysteine ligands. Details of the electronic structure of the Ni(III) site available from the DFT calculations point to subtle changes in the unpaired spin density on the S-donors as being responsible for the altered sensitivity of Ala0-NiSOD to O2.

Crystal structure of a soluble fragment of poliovirus 2CATPase.

Poliovirus (PV) 2CATPase is the most studied 2C protein in the Picornaviridae family. It is involved in RNA replication, encapsidation and uncoating and many inhibitors have been found that target PV 2CATPase. Despite numerous investigations to characterize its functions, a high-resolution structure of PV 2C has not yet been determined. We report here the crystal structure of a soluble fragment of PV 2CATPase to 2.55Å, containing an ATPase domain, a zinc finger and a C-terminal helical domain but missing the N-terminal domain. The ATPase domain shares the common structural features with EV71 2C and other Superfamily 3 helicases. The C-terminal cysteine-rich motif folds into a CCCC type zinc finger in which four cysteine ligands and several auxiliary residues assist in zinc binding. By comparing with the known zinc finger fold groups, we found the zinc finger of 2C proteins belong to a new fold group, which we denote the “Enterovirus 2C-like” group. The C-terminus of PV 2CATPase forms an amphipathic helix that occupies a hydrophobic pocket located on an adjacent PV 2CATPase in the crystal lattice. The C-terminus mediated PV 2C-2C interaction promotes self-oligomerization, most likely hexamerization, which is fundamental to the ATPase activity of 2C. The zinc finger is the most structurally diverse feature in 2C proteins. Available structural and virological data suggest that the zinc finger of 2C might confer the specificity of interaction with other proteins. We built a hexameric ring model of PV 2CATPase and visualized the previously identified functional motifs and drug-resistant sites, thus providing a structure framework for antiviral drug development.

Heme FeSO2− intermediates in sulfite reduction: Contrasts with FeOO2− species from oxygen–oxygen bond activating systems

AbstractSulfite reductase (SiR) catalyzes a six electron and six proton reduction of sulfite to sulfide. Similarly to the cytochrome P450 (cytP450) family, the active site in SiR contains a (partially reduced) heme bound axially to a cysteinate ligand—though with an extra Fe4S4 cluster. Fe(III)SO2−, Fe(III)SOH−, and Fe(III)SO(H2) intermediates have been proposed for the catalytic cycle of SiR, leading to a formally Fe(V)S species—akin to the widely accepted reaction mechanism in cytP450. Here, density functional theory (DFT) data is reported for of such FeSO(H2) intermediates. The Fe(III)SO2− models display relatively high energies for homolytic bond breaking compared to their isomeric oxygen‐bound Fe(III)OS2− models, and thus offer a better alternative in terms of avoiding radical side products able to induce enzyme suicide. This could be due to the fact that the (iron‐bound) sulfur is more active from a redox standpoint compared to oxygen, thus permitting the departing oxygen to maintain a redox‐inert state. Di‐protonation of the oxygen is computed to lead to a compound I type Fe(IV)S coupled to a porphyrin radical anion—consistent with an intermediate previously observed by x‐ray crystallography.

A novel cysteine-linked antibacterial surface coating significantly inhibits bacterial colonization of nasal silicone prongs in a phase one pre-clinical trial

Ventilator associated pneumonia and sepsis are frequent complications in neonatal care. Bacterial colonization of medical devices and interfaces used for respiratory support may contribute by functioning as a bacterial reservoir seeding bacteria into airways. We have developed an antibacterial surface coating based on a cysteine ligand covalently coupled via a spacer to a carboxylic backbone layer on an acrylic acid grafted silicone surface. This coating was applied on a commercially available nasal prong and the antibacterial effect was evaluated both in vitro and in vivo in a first-in-human phase 1 trial. The coated nasal prongs had strong antibacterial activity against both Gram-negative and Gram-positive bacteria in vitro. In a randomized pre-clinical trial study of 24 + 24 healthy adult volunteers who carried coated or non-coated nasal prongs for 18 h, a 10log difference in mean bacterial colonization of 5.82 (p < 0.0001) was observed. These results show that this coating technique can prevent colonization by the normal skin and mucosal flora, and thus represent a promising novel technology for reduction of medical device-associated hospital acquired infections.

Preparation and Characterization of Chiral Transition-Metal Dichalcogenide Quantum Dots and Their Enantioselective Catalysis.

Two-dimensional transition-metal dichalcogenides (TMDs) had attracted enormous interests owing to their extraordinary optical, physical, and chemical properties. Herein, we prepared for the first time a series of chiral TMD quantum dots (QDs) from MoS2 and WS2 bulk crystals by covalent modification with chiral ligands cysteine and penicillamine. The chiral TMD QDs were carefully investigated by spectroscopic and microscopic techniques. Their chiral optical activity was confirmed by distinct circular dichroism signals different to those of the chiral ligands. Interestingly, with the assistance of copper ions, the chiral QDs displayed strong and chiral selective peroxidase-like activity. Up to now, inorganic nanomaterials with peroxidase-like activity were tremendous but seldom examples with enantioselectivity. The enantioselectivity of our chiral TMD QDs toward chiral substrates d- and l-tyrosinol was highly up to 6.77, which was almost the best performance ever reported. The mechanisms of enantioselectivity was further investigated by quartz crystal microbalance assays. We believed that because of the extraordinary electronic and optical properties, the chiral TMD QDs should be useful for nonlinear optical materials, asymmetric catalysis, chiral and biological sensors, and so on.

Mitochondrial complex I in the post-ischemic heart: reperfusion-mediated oxidative injury and protein cysteine sulfonation

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O2−. The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron‑sulfur clusters, including N3 (C425), N1b (C92), N4 (C226), N2 (C158/C188), and N1a (C134/C139). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C134/C139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O2− generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C125, C187, C206, C238, C255, C286), 75 kDa (C367, C554, C564, C727), 49 kDa (C146, C326, C347), and PSST (C188). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C67, C112, C183, C253), 15 kDa (C43), and 13 kDa (C79). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C43 of the 15 kDa subunit forms an intramolecular disulfide bond with C56, which was reported to stabilize complex I structure. C79 of the 13 kDa subunit is involved in Zn2+-binding, which was reported functionally important for complex I assembly. C79 sulfonation by I/R was found to impair Zn2+-binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing •O2− production, and mediating redox dysfunction of complex I.

Increased Protein Cysteine Sulfonation with Heme Destruction of Mitochondrial Complex III Mediates Cardiac Reperfusion Injury

A serious consequence of myocardial ischemia‐reperfusion injury (IR) is oxidative damage, which causes mitochondrial dysfunction. Such IR induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide. The cascading reactive oxygen species can propagate and potentially induce protein cysteine sulfonation (PrCSO3H) of mitochondrial proteins. Herein we employed UV/VIS spectroscopy and LC‐MS/MS analysis to study the mechanism of IR‐mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30 min of ischemia (coronary ligation) and 24 h of reperfusion in vivo. In the IR region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p&lt;0.001). Visible spectroscopic analysis indicated that IR mediated destruction of heme b and hemes (c+c1) in the mitochondria, thus supporting IR‐induced impairment of the enzymatic activity of complex III. However, no significant impairment of complex III activity and heme destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min coronary ligation in vivo despite a decreased state 3 respiration detected in the ischemic mitochondria. LC‐MS/MS analysis showed formation of PrCSO3H, which was consistent with a redox analysis of mitochondria following IR (EPR measurement of oxidation of cyclic hydroxylamine). Mitochondria from IR regions had intensely increased oxidative modification with S‐sulfonation level on the cysteine ligand (at C236) of 2Fe‐2S cluster of Rieske iron‐sulfur protein (uqcrfs1), thus impairing the main electron transfer pathway controlled by the Q‐cycle mechanism of complex III. LC‐MS/MS analysis also indicated that IR‐mediated increased PrCSO3H of hinge protein at C65 (subunit 8, uqcrh) and cytochrome c1 subunit at C140 and C220, which weakened the interaction of hinge protein with cytochrome c1 and proper complex formation between cytochrome c1 and cytochrome c. LC‐MS/MS analysis further identified 4‐HNE (4‐hydroxylnonenal) adduct of cytochrome c1 at C140, suggesting involvement of IR‐induced lipid peroxidation in heme destruction and PrCSO3H of complex III. LC‐MS/MS analysis also showed that IR‐enhancing PrCSO3H of core 1 subunit (uqcrc1 at C69, C268, C380, C410, C445, C453) and core 2 subunit (uqcrc2 at C191), which may affect the binding of subunit 9 inhibitory peptide to core 1 and core 2 subunits and reactivate the MPP (matrix processing peptidase) activity of the core 1/core 2 subunits of mammalian complex III. LC‐MS/MS analysis of ischemic mitochondria has indicated a modest reduction from the basal level of complex III PrCSO3H detected in the mitochondria of sham control heart, suggesting that physiologic conditions of hypoxia during ischemia suppressed PrCSO3H, and physiologic conditions of hyperoxygenation during reperfusion mediated enhancement of complex III PrCSO3H. In conclusion, reperfusion‐mediated heme destruction with increased PrCSO3H controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction in the post‐ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may related to the preservation of functional integrity of heme groups and protein structure within mitochondrial complex III.Support or Funding InformationNHLBI/NIH, HL083237This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ferredoxin: the central hub connecting photosystem I to cellular metabolism

Ferredoxin (Fd) is a small soluble iron-sulfur protein essential in almost all oxygenic photosynthetic organisms. It contains a single [2Fe-2S] cluster coordinated by four cysteine ligands. It accepts electrons from the stromal surface of PSI and facilitates transfer to a myriad of acceptors involved in diverse metabolic processes, including generation of NADPH via Fd-NADP-reductase, cyclic electron transport for ATP synthesis, nitrate reduction, nitrite reductase, sulfite reduction, hydrogenase and other reductive reactions. Fd serves as the central hub for these diverse cellular reactions and is integral to complex cellular metabolic networks. We describe advances on the central role of Fd and its evolutionary role from cyanobacteria to algae/plants. We compare structural diversity of Fd partners to understand this orchestrating role and shed light on how Fd dynamically partitions between competing partner proteins to enable the optimum transfer of PSI-derived electrons to support cell growth and metabolism.

Synthesis and Characterization of Aqueous Lead Selenide Quantum Dots for Solar Cell Application

High quality, colloidal lead selenide (PbSe) nanoparticles possessing cube shaped morphology have been successfully synthesized by organometallic synthesis method, using oleic acid (OA) as capping agent. The use of non-coordinating solvent, 1-Octadecene (ODE), during the synthesis results in good quality nanocrystals. Morphology analysis by transmission electron microscopy reveals that cube-shaped nanocrystals with a size range of 10 nm have been produced during the synthesis. The absorption and PL spectra analysis showed an emission peak at 675 nm when excited to a wavelength of 610 nm, further confirmed the formation of PbSe nanocrystals. The surface modification of this colloidal quantum dots was then carried out using L- cysteine ligand, to make them water soluble, for solar cell application. The J-V characteristics study of this PbSe quantum dots solar cell (PbSe QDSC) showed a little power conversion efficiency which intern it shows significant advance toward effective utilization of PbSe nanocrystals sensitized in solar cells.

Influence of Conserved Structural Elements of the Proximal Pocket in HEME-Thiolate Enzymes on Oxygen Insertion Reactions

In many heme-thiolate enzymes, the chemical transformations of substrates occur in a binding site located on the distal side of the heme. The opposite (proximal) side of the heme is characterized by conserved structural elements that vary subtly from enzyme to enzyme. These structural elements include hydrogen bonds to the proximal sulfur and the dipole moment of an alpha helix that terminates in the proximal cysteine ligand. We present research on two heme-thiolate enzymes, chloroperoxidase and cytochrome P450CAM, that shows that these structural elements have significant influence on a wide range of reactivities (formation of compound I, epoxidation and allylic hydroxylation of alkene substrates, regioselectivity and even enantiospecificity). The fine-tuning of these structural motifs can select among closely related modes of reactivity.

Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S] Cluster Ligands by 13Cβ-Cysteine Labeling.

Iron-sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4-n (n = 0, 1, 2) cluster by selective cysteine-13Cβ isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-13Cβ hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

157 - Increased Cysteine Sulfonation of the Hydrophilic Domain of Complex I Mediates Mitochondrial Dysfunction in the Post-ischemic Heart

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration, decreased ATP generation, and overproduction of O2. The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to myocardial injury. Herein we employed LC-MS/MS to identify protein cysteine oxidation of complex I in the mitochondria of sham control rat hearts and I/R mitochondria from the infarct region of rat hearts subjected to 30 min of coronary ligation and 24h of reperfusion in vivo. Mitochondrial preparations from infarct regions had significantly enhanced S-sulfonation (C-SO3) levels on the cysteine ligands of iron-sulfur clusters, including N3 (C425), N1b (C92), N4 (C226), N2 (C158/C188), and N1a (C134/C139). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus C-SO of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and functions as temporary storage for electrons and antioxidants. Increased C134/C139 C-SO by I/R impaired the cluster N1a, increasing O2 generation by complex I via FMNH∙ autooxidation. LC-MS/MS analysis also revealed I/R-mediated increased C-SO of regulatory thiols at the core subunits of Ndufv1 (C125, C142, C187, C206, C238, C255, C286), Ndus1 (C367, C554, C564, C727), and Ndufs2 (C146, C326, C347). These results were consistent with the consensus indicating that Ndufv1 and Ndus1 are two major polypeptides hosting regulatory thiols, and their enhanced C-SO by I/R predisposed the post-ischemic myocardium to have a more oxidized physiological setting. LC-MS/MS analysis further showed I/R-mediated enhanced C-SO at the supernumerary subunits of Ndufa10 (C67, C112, C183, C253), Ndufs5 (C43), and Ndufs6 (C79). The Ndufa10 subunit (42 kDa) is metazoan-specific, and loosely bound to complex I with a functional implication of stabilizing mammalian complex I. I/R-mediated enhanced C-SO of Ndufs10 weakens docking Ndufa10 subunit to the ND2 subunit, and destabilizes the structure of complex I. The Ndufs5 subunit (15 kDa) is confined to the face of intermembrane space. C43 of Ndufs5 is involved in twin CX9C motifs (C33/C43 and C56/C66), and forms an intramolecular disulfide bond with C56, which is functionally important for complex I stability. I/R-mediated C43 C-SO thus destabilized complex I; it is a consequence of reductively breaking the disulfide bond by ischemia followed by reperfusion-induced cysteine oxidation. C79 of Ndufs6 is involved in Zn2+-binding, which is functionally important for complex I assembly. C79 C-SO by I/R impaired Zn2+-binding, thus blocking a late step of complex I assembly and biogenesis. In conclusion, protein sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increases O2 production and pro-oxidant activity, and impairs the stability, assembly, and biogenesis of mammalian complex I.

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN−) ligands. Hydrogen (H2) and oxygen (O2) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. 57Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fed in a [4Fe4S-2Fe]3− complex with the net spin on Fed whereas Hox-CO shows an apical CN− at Fed in a [4Fe4S-2Fe(CO)]3− complex with net spin sharing among Fep and Fed (proximal or distal iron ions in [2Fe]). At ambient O2 pressure, a novel H-cluster species (Hox-O2) accumulated in C169A, assigned to a [4Fe4S-2Fe(O2)]3− complex with an apical superoxide (O2−) carrying the net spin bound at Fed. H2 exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]3− complex with the net spin on the reduced cubane, an apical hydride at Fed, and a proton at a cysteine ligand. Hox-O2 and Hhyd are stabilized by impaired O2– protonation or proton release after H2 cleavage due to interruption of the proton path towards and out of the active site.

Screening of anionic-modified polymers in terms of stability, disintegration, and swelling behavior

This study aimed to screen the stability, disintegration, and swelling behavior of chemically modified anionic polymers. Investigated polymers were well-known and widely used staples of the pharmaceutical and medical field, namely, alginate (AL), carboxymethyl cellulose (CMC), polycarbophil (PC), and hyaluronic acid (HA). On the basis of amide bond formation between the carboxylic acid moieties of anionic polymers and the primary amino group of the modification ligand cysteine (CYS), the modified polymers were obtained. Unmodified polymers served as controls throughout all studies. With the Ellman’s assay, modification degrees were determined of synthesized polymeric excipients. Stability assay in terms of erosion study at physiological conditions were performed. Moreover, water uptake of compressed polymeric discs were evaluated and further disintegration studies according to the USP were carried out to define the potential ranking. Results ranking figured out PCCYS > CMCCYS > HACYS > ALCYS in terms of water uptake capacity compared to respective controls. Cell viability assays on Caco-2 cell line as well as on RPMI 2650 (ATTC CCL30) proved modification not being harmful to those. Due to the results of this study, an intense screening of prominent anionic polymer derivate was performed in order to help the pharmaceutical research for the best choice of polymeric excipients for developments of controlled drug release systems.

Heme–thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.

Thiolate Spin Population of Type I Copper in Azurin Derived from 33S Hyperfine Coupling.

The electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper-sulfur (Cu-S) bond. The structure and electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired electron to neighboring ligands provides an elegant experimental way to probe the fine details of the electronic structure of type I copper. To date, the crucial parameter of electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared 33S-enriched azurin and carried out W-band (95 GHz) electron paramagnetic resonance (EPR) and electron-electron double resonance detected NMR (EDNMR) measurements and, for the first time, recorded the 33S nuclear frequencies, from which the hyperfine coupling and the spin population on the sulfur of the thiolate ligand were derived. The overlapping 33S and 14N EDNMR signals were resolved using a recently introduced two-dimensional correlation technique, 2D-EDNMR. The 33S hyperfine tensor was determined by simulations of the EDNMR spectra using 33S hyperfine and quadrupolar tensors predicted by QM/MM DFT calculations as starting points for a manual spectral fit procedure. To reach a reasonable agreement with the experimental spectra, the 33S hyperfine principal value, Az, and one of the corresponding Euler angles had to be modified. The final values obtained gave an experimentally determined sulfur spin population of 29.8 ± 0.7%, significantly improving the wide range of 29-62% reported in the literature. Our direct, experimentally derived value now provides an important constraint for further theoretical work aimed at unravelling the unique electronic properties of this site.