Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography.

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

Enhancing the Carbon Monoxide Oxidation Performance through Surface Defect Enrichment of Ceria-Based Supports for Platinum Catalyst.

Effective synthesis and application of single-atom catalysts on supports lacking enough defects remain a significant challenge in environmental catalysis. Herein, we present a universal defect-enrichment strategy to increase the surface defects of CeO2-based supports through H2 reduction pretreatment. The Pt catalysts supported by defective CeO2-based supports, including CeO2, CeZrOx, and CeO2/Al2O3 (CA), exhibit much higher Pt dispersion and CO oxidation activity upon reduction activation compared to their counterpart catalysts without defect enrichment. Specifically, Pt is present as embedded single atoms on the CA support with enriched surface defects (CA-HD) based on which the highly active catalyst showing embedded Pt clusters (PtC) with the bottom layer of Pt atoms substituting the Ce cations in the CeO2 surface lattice can be obtained through reduction activation. Embedded PtC can better facilitate CO adsorption and promote O2 activation at PtC-CeO2 interfaces, thereby contributing to the superior low-temperature CO oxidation activity of the Pt/CA-HD catalyst after activation.

Endogenous and exogeneous stimuli-triggered reactive oxygen species evoke long-lived carbon monoxide to fight against lung cancer.

Reactive oxygen species (ROS)-associated anticancer approaches usually suffer from two limitations, i.e., insufficient ROS level and short ROS half-life. Nevertheless, no report has synchronously addressed both concerns yet. Herein, a multichannel actions-enabled nanotherapeutic platform using hollow manganese dioxide (H-MnO2) carriers to load chlorin e6 (Ce6) sonosensitizer and CO donor (e.g., Mn2(CO)10) has been constructed to maximumly elevate ROS level and trigger cascade catalysis to produce CO. Therein, intratumoral H2O2 and ultrasound as endogenous and exogeneous triggers stimulate H-MnO2 and Ce6 to produce •OH and 1O2, respectively. The further cascade reaction between ROS and Mn2(CO)10 proceeds to release CO, converting short-lived ROS into long-lived CO. Contributed by them, such a maximumly-elevated ROS accumulation and long-lived CO release successfully suppresses the progression, recurrence and metastasis of lung cancer with a prolonged survival rate. More significantly, proteomic and genomic investigations uncover that the CO-induced activation of AKT signaling pathway, NRF-2 phosphorylation and HMOX-1 overexpression induce mitochondrial dysfunction to boost anti-tumor consequences. Thus, this cascade catalysis strategy can behave as a general means to enrich ROS and trigger CO release against refractory cancers.

Intensified bioleaching of a spent Co-Mo catalyst through the addition of extracellular polymeric substances (EPSs) and its mechanism exploration.

The extraction and recovery of valuable metals from various spent catalysts via bioleaching represents a green, low-carbon and eco-friendly process. However, the pulp density of spent catalysts is usually 1.0% or lower owing to their toxicity, denoting low process capacity and poor practical potential. In this study, an intensified bioleaching strategy was used for the first time to promote the release efficiencies of both Co and Mo from a spent Co-Mo catalyst at a high pulp density of 10% by supplementing extracellular polymeric substances (EPSs). The results showed that the addition of 0.6 g L-1 EPSs harvested a maximum release of 73.6% for Co and 72.5% for Mo after 9 days of contact, with an evident elevation of 22.6% for Co and 24.4% for Mo, in contrast to no addition, respectively. The added EPS not only promoted the growth of plankton cells to produce more active molecules but also boosted the adhesion of leaching cells to the spent catalyst to form stable aggregates. Moreover, the resulting aggregates allowed for the gathering and confinement of the active small molecules, including Fe3+ and Fe2+, inside the micro-areas between the spent catalysts and the cells for quick electronic transfer as an interface oxidation/reduction reaction to free both Co and Mo from the spent catalyst.

Insights into the photoactivatable CO releasing properties of dicarbonyl Ru(II) complex with 8-amino quinoline ligand: Experimental and theoretical studies.

Reaction between the polymeric [RuCl2(CO)2]n and the N,N-bidentate ligand, 8-amino-quinoline (Quin), in methanol, afforded the photoactivated CO releasing molecule with the formula of trans-(Cl,Cl)-[RuCl2(CO)2Quin]. In the presence of biomolecules or in solvents with varying polarity and coordinating abilities, the solvatochromic characteristics and dark stability were investigated. A new board band emerged in the visible spectrum during the illumination, and its position varies according to the type of solvent used, indicating the role of the solvent in controlling the nature of the CO-depleted species. Spectral methods were used in combination with density functional theory simulations to get insight into the local minimum structure and the electronic properties of the Ru(II) complex. The results of the myoglobin assay showed that within the first two hours of illumination, one of the two CO molecules was released. The cytotoxic properties of the Ru(II)-based complex were investigated against normal mice bone marrow stromal cells and malignant human acute monocytic leukaemia cells.

Antimicrobial and anticancer properties of carbon monoxide releasing molecules of the fac-[Re(CO)3(N-N)L]+ family.

The toxicity profile of fac-[Re(CO)3(N-N)L]+ complexes against microbial and tumoral cells has been extensively studied, primarily focusing on modifications to the bidentate diimine (N-N) ligand. However, less attention has been paid to modifications of the axial ligand L, which is perpendicular to the Re-N-N plane. This study reveals that the high toxicity of the fac-[Re(CO)3(bpy)(Ctz)]+ complex may be attributed to the structural effect of the trityl (CPh3) group present in clotrimazole, as removal of phenyl rings causes a significant decrease in the activity against Staphylococcus aureus (S. aureus). Moreover, substitution of the 1-tritylimidazole ligand by the structurally related ligands PPh3 and PCy3 maintains similarly high activity levels. These findings contribute to understanding the interactions of toxic complexes with bacterial membranes, suggesting that the ligand structures play a crucial role in inhibiting cell wall synthesis processes, potentially including Lipid II synthesis. Compounds with Ph3E (E = C-imidazole; P) groups also showed to be 10 times more toxic than cisplatin against three mammalian cell lines (IC50: 2-4 µM). In contrast, the analogue 1-benzylimidazole and 1-tert-butylimidazole derivatives were as toxic as cisplatin. We observed that the decomposition of the [Re(I)(CO)3] fragment inside mammalian cell lines liberates CO, which is expected to exert biological effects. Therefore, compounds of this family possessing the structural motif Ph3E seem to combine high antimicrobial and antitumoral activities, the latter being much higher than that of cisplatin.

Unraveling the Synergistic Mechanism of Ir Species with Various Electron Densities over an Ir/ZSM-5 Catalyst Enables High-Efficiency NO Reduction by CO.

Selective catalytic reduction using CO as a reducing agent (CO-SCR) has exhibited its application potential in coal-fired, steel, and other industrial sectors. In comparison to NH3-SCR, CO-SCR can achieve synergistic control of CO and NO pollutants, making it a powerful denitrification technology that treats waste with waste. Unfortunately, the competitive adsorption of O2 and NO on CO-SCR catalysts inhibits efficient conversion of NOx under O2-containing conditions. In this work, we obtained two Ir sites with different electron densities, Ir1 single atoms in the oxidized Irδ+ state and Ir0 nanoparticles in the metallic state, by controlled pretreatment of the Ir/ZSM-5 catalyst with H2 at 200 °C. The coexistence of Ir1 single atoms and Ir0 nanoparticles on ZSM-5 creates a synergistic effect, which facilitates the reduction of NO through CO in the presence of O2, following the Langmuir-Hinshelwood mechanism. The ONNO dimer is formed on the Ir1 single atom sites and then spills over to the neighboring Ir0 nanoparticles for subsequent reduction to N2 by CO. Specifically, this tandem reaction enables 83% NO conversion and 100% CO conversion on an Ir-based catalyst at 250 °C under 3% O2.

IrSn Bimetallic Clusters Confined in MFI Zeolites for CO Selective Catalytic Reduction of NOx in the Presence of Excess O2.

We developed a simple strategy for preparing IrSn bimetallic clusters encapsulated in pure silicon zeolites via a one-pot hydrothermal synthesis by using diethylamine as a stabilizing agent. A series of investigations verified that metal species have been confined successfully in the inner of MFI zeolites. IrSn bimetallic cluster catalysts were efficient for the CO selective catalytic reduction of NOx in the presence of excess O2. Furthermore, the 13CO temperature-programmed surface reaction results demonstrated that NO2 and N2O could form when most of the CO was transformed into CO2 and that Sn modification could passivate CO oxidation on the IrSn bimetallic clusters, leading to more reductants that could be used for NOx reduction at high temperatures. Furthermore, SO2 can also influence the NOx conversion by inhibiting the oxidation of CO. This study provides a new strategy for preparing efficient environmental catalysts with a high dispersion of metal species.

Gaseous inhibition of the transsulfuration pathway by cystathionine ß-synthase.

The transsulfuration pathway plays a key role in mammals for maintaining the balance between cysteine and homocysteine, whose concentrations are critical in several biochemical processes. Human cystathionine ß-synthase is a heme-containing, pyridoxal 5'-phosphate (PLP)-dependent enzyme found in this pathway. The heme group does not participate directly in catalysis, but has a regulatory function, whereby CO or NO binding inhibits the PLP-dependent reactions. In this study, we explore the detailed structural changes responsible for inhibition using quantum chemical calculations to validate the experimentally observed bonding patterns associated with heme CO and NO binding and molecular dynamics simulations to explore the medium-range structural changes triggered by gas binding and propagating to the PLP active site, which is more than 20 Å distant from the heme group. Our results support a previously proposed mechanical signaling model, whereby the cysteine decoordination associated with gas ligand binding leads to breaking of a hydrogen bond with an arginine residue on a neighbouring helix. In turn, this leads to a shift in position of the helix, and hence also of the PLP cofactor, ultimately disrupting a key hydrogen bond that stabilizes the PLP in its catalytically active form.

Enhanced Catalytic Oxidation Reactivity over Atomically Dispersed Pt/CeO2 Catalysts by CO Activation.

The elevation of the low-temperature oxidation activity for Pt/CeO2 catalysts is challenging to meet the increasingly stringent requirements for effectively eliminating carbon monoxide (CO) from automobile exhaust. Although reducing activation is a facile strategy for boosting reactivity, past research has mainly concentrated on applying H2 as the reductant, ignoring the reduction capabilities of CO itself, a prevalent component of automobile exhaust. Herein, atomically dispersed Pt/CeO2 was fabricated and activated by CO, which could lower the 90% conversion temperature (T90) by 256 °C and achieve a 20-fold higher CO consumption rate at 200 °C. The activated Pt/CeO2 catalysts showed exceptional catalytic oxidation activity and robust hydrothermal stability under the simulated working conditions for gasoline or diesel exhausts. Characterization results illustrated that the CO activation triggered the formation of a large portion of Pt0 terrace sites, acting as inherent active sites for CO oxidation. Besides, CO activation weakened the Pt-O-Ce bond strength to generate a surface oxygen vacancy (Vo). It served as the oxygen reservoir to store the dissociated oxygen and convert it into active dioxygen intermediates. Conversely, H2 activation failed to stimulate Vo, but triggered a deactivating transformation of the Pt nanocluster into inactive PtxOy in the presence of oxygen. The present work offers coherent insight into the upsurging effect of CO activation on Pt/CeO2, aiming to set up a valuable avenue in elevating the efficiency of eliminating CO, C3H6, and NH3 from automobile exhaust.

Polyprodrug nanomedicine for chemiexcitation-triggered self-augmented cancer chemotherapy and gas therapy.

Carbon monoxide (CO) has emerged as a potential antitumor agent by inducing the dysfunction of mitochondria and the apoptosis of cancer cells. However, it remains challenging to deliver appropriate amount of CO into tumor to ensure efficient tumor growth suppression with minimum side effects. Herein we developed a CO prodrug-loaded nanomedicine based on the self-assembly of camptothecin (CPT) polyprodrug amphiphiles. The polyprodrug nanoparticles readily dissociate upon exposure to endogenous H2O2 in the tumor, resulting in rapid release of CPT and generation of high-energy intermediate dioxetanedione. The latter can transfer the energy to neighboring CO prodrugs to activate CO production by chemiexcitation, while CPT promotes the generation of H2O2 in tumors, which in turn facilitates cascade CPT and CO release. As a result, the polyprodrug nanoparticles display remarkable tumor suppression in both subcutaneous and orthotopic breast tumor-bearing mice owing to the self-augmented CPT release and CO generation. In addition, no obvious systemic toxicity was observed in mice treated with the metal-free CO prodrug-loaded nanomedicine, suggesting the good biocompatibility of the polyprodrug nanoparticles. Our work provides new insights into the design and construction of polyprodrug nanomedicines for synergistic chemo/gas therapy.

Metal-based carbon monoxide releasing molecules with promising cytotoxic properties.

Carbon monoxide, the "silent killer" gas, is increasingly recognised as an important signalling molecule in human physiology, which has beneficial biological properties. A particular way of achieving controlled CO administration is based on the use of biocompatible molecules that only release CO when triggered by internal or external factors. These approaches include the development of pharmacologically effective prodrugs known as CO releasing molecules (CORMs), which can supply biological systems with CO in well-regulated doses. An overview of transition metal-based CORMs with cytotoxic properties is here reported. The mechanisms at the basis of the biological activities of these molecules and their potential therapeutical applications with respect to their stability and CO releasing properties have been discussed. The activation of metal-based CORMs is determined by the type of metal and by the nature and features of the auxiliary ligands, which affect the metal core electronic density and therefore the prodrug resistance towards oxidation and CO release ability. A major role in regulating the cytotoxic properties of these CORMs is played by CO and/or CO-depleted species. However, several mysteries concerning the cytotoxicity of CORMs remain as intriguing questions for scientists.

Fluorescent small molecule donors.

Small molecule donors (SMDs) play subtle roles in the signaling mechanism and disease treatments. While many excellent SMDs have been developed, dosage control, targeted delivery, spatiotemporal feedback, as well as the efficiency evaluation of small molecules are still key challenges. Accordingly, fluorescent small molecule donors (FSMDs) have emerged to meet these challenges. FSMDs enable controllable release and non-invasive real-time monitoring, providing significant advantages for drug development and clinical diagnosis. Integration of FSMDs with chemotherapeutic, photodynamic or photothermal properties can take full advantage of each mode to enhance therapeutic efficacy. Given the remarkable properties and the thriving development of FSMDs, we believe a review is needed to summarize the design, triggering strategies and tracking mechanisms of FSMDs. With this review, we compiled FSMDs for most small molecules (nitric oxide, carbon monoxide, hydrogen sulfide, sulfur dioxide, reactive oxygen species and formaldehyde), and discuss recent progress concerning their molecular design, structural classification, mechanisms of generation, triggered release, structure-activity relationships, and the fluorescence response mechanism. Firstly, from the large number of fluorescent small molecular donors available, we have organized the common structures for producing different types of small molecules, providing a general strategy for the development of FSMDs. Secondly, we have classified FSMDs in terms of the respective donor types and fluorophore structures. Thirdly, we discuss the mechanisms and factors associated with the controlled release of small molecules and the regulation of the fluorescence responses, from which universal guidelines for optical properties and structure rearrangement were established, mainly involving light-controlled, enzyme-activated, reactive oxygen species-triggered, biothiol-triggered, single-electron reduction, click chemistry, and other triggering mechanisms. Fourthly, representative applications of FSMDs for trackable release, and evaluation monitoring, as well as for visible in vivo treatment are outlined, to illustrate the potential of FSMDs in drug screening and precision medicine. Finally, we discuss the opportunities and remaining challenges for the development of FSMDs for practical and clinical applications, which we anticipate will stimulate the attention of researchers in the diverse fields of chemistry, pharmacology, chemical biology and clinical chemistry. With this review, we hope to impart new understanding thereby enabling the rapid development of the next generation of FSMDs.

A CO-releasing coating based on carboxymethyl chitosan-functionalized graphene oxide for improving the anticorrosion and biocompatibility of magnesium alloy stent materials.

Due to its biofunctions similar to NO, the CO gas signaling molecule has gradually shown great potential in cardiovascular biomaterials for regulating the in vivo performances after the implantation and has received increasing attention. To construct a bioactive surface with CO-releasing properties on the surface of magnesium-based alloy to augment the anticorrosion and biocompatibility, graphene oxide (GO) was firstly modified using carboxymethyl chitosan (CS), and then CO-releasing molecules (CORM401) were introduced to synthesize a novel biocompatible nanomaterial (GOCS-CO) that can release CO in the physiological environments. The GOCS-CO was further immobilized on the magnesium alloy surface modified by polydopamine coating with Zn2+ (PDA/Zn) to create a bioactive surface capable of releasing CO in the physiological environment. The outcomes showed that the CO-releasing coating can not only significantly enhance the anticorrosion and abate the corrosion degradation rate of the magnesium alloy in a simulated physiological environment, but also endow it with good hydrophilicity and a certain ability to adsorb albumin selectively. Owing to the significant enhancement of anticorrosion and hydrophilicity, coupled with the bioactivity of GOCS, the modified sample not only showed excellent ability to prevent platelet adhesion and activation and reduce hemolysis rate but also can promote endothelial cell (EC) adhesion, proliferation as well as the expression of nitric oxide (NO) and vascular endothelial growth factor (VEGF). In the case of CO release, the hemocompatibility and EC growth behaviors were further significantly improved, suggesting that CO molecules released from the surface can significantly improve the hemocompatibility and EC growth. Consequently, the present study provides a novel surface modification method that can simultaneously augment the anticorrosion and biocompatibility of magnesium-based alloys, which will strongly promote the research and application of CO-releasing bioactive coatings for surface functionalization of cardiovascular biomaterials and devices.

Gases and gas-releasing materials for the treatment of chronic diabetic wounds.

Chronic non-healing wounds are a common consequence of skin ulceration in diabetic patients, with severe cases such as diabetic foot even leading to amputations. The interplay between pathological factors like hypoxia-ischemia, chronic inflammation, bacterial infection, impaired angiogenesis, and accumulation of advanced glycosylation end products (AGEs), resulting from the dysregulation of the immune microenvironment caused by hyperglycemia, establishes an unending cycle that hampers wound healing. However, there remains a dearth of sufficient and effective approaches to break this vicious cycle within the complex immune microenvironment. Consequently, numerous scholars have directed their research efforts towards addressing chronic diabetic wound repair. In recent years, gases including Oxygen (O2), Nitric oxide (NO), Hydrogen (H2), Hydrogen sulfide (H2S), Ozone (O3), Carbon monoxide (CO) and Nitrous oxide (N2O), along with gas-releasing materials associated with them have emerged as promising therapeutic solutions due to their ability to regulate angiogenesis, intracellular oxygenation levels, exhibit antibacterial and anti-inflammatory effects while effectively minimizing drug residue-induced damage and circumventing drug resistance issues. In this review, we discuss the latest advances in the mechanisms of action and treatment of these gases and related gas-releasing materials in diabetic wound repair. We hope that this review can provide different ideas for the future design and application of gas therapy for chronic diabetic wounds.

Dual delivery of carbon monoxide and doxorubicin using haemoglobin-albumin cluster: proof of concept for well-tolerated cancer therapy.

A serious concern of doxorubicin (DOX) therapy is that it causes severe adverse effects, particularly cardiotoxicity. Carbon monoxide (CO) possesses powerful cytoprotective effects against drug-induced organ injury and is expected to ameliorate DOX-induced cardiotoxicity. In this study, a dual carrier of DOX and CO (CO-HemoAct-DOX) was fabricated based on a haemoglobin-albumin cluster (HemoAct), which is a protein cluster with a haemoglobin core structure wrapped by serum albumin. CO-HemoAct-DOX was synthesised by binding CO to a haemoglobin core and covalently conjugating (6-maleimidocaproyl)hydrazone derivative of DOX to an albumin shell. The average DOX/cluster ratio was about 2.6. In the in vitro cytotoxicity assay against cancer cells, the anti-tumour activity of CO-HemoAct-DOX was 10-fold lower than that of DOX in a 2D-cultured model, whereas CO-HemoAct-DOX suppressed the growth of tumour spheroids to the same extent as DOX in the 3D-cultured model. In colon-26 tumour-bearing mice, CO-HemoAct-DOX achieved DOX delivery to the tumour site and alleviated tumour growth more effectively than DOX. Furthermore, CO-HemoAct attenuated DOX-induced cardiomyocyte atrophy in H9c2 cells and elevated the levels of cardiac biomarkers in mice exposed to DOX. These results suggest that the dual delivery of CO and DOX using HemoAct is a promising strategy as an anti-tumour agent to realise well-tolerated cancer therapy with minimal cardiotoxicity.

Cascade Self-Generation of Carbon Monoxide Triggered by Photoinduced Holes for Efficient Hypoxic Tumors Therapy.

It still remains challenging to design multifunctional therapeutic reagents for effective cancer therapy under a unique tumor microenvironment including insufficient endogenous H2O2 and O2, low pH, and a high concentration of glutathione (GSH). In this work, a CO-based phototherapeutic system triggered by photogenerated holes, which consisted of ionic liquid (IL), the CO prodrug Mn2(CO)10, and iridium(III) porphyrin (IrPor) modified carbonized ZIF-8-doped graphitic carbon nitride nanocomposite (IL/ZCN@Ir(CO)), was designed for cascade hypoxic tumors. Upon light irradiation, the photogenerated holes on IL/ZCN@Ir(CO) oxidize water into H2O2, which subsequently induces Mn2(CO)10 to release CO. Meanwhile, IrPor can convert H2O2 to hydroxyl radical (•OH) and subsequent singlet oxygen (1O2), which further triggers CO release. Moreover, the degraded MnO2 shows activity for glutathione (GSH) depletion and mimics peroxidase, leading to GSH reduction and •OH production in tumors. Thus, this strategy can in situ release high concentrations of CO and reactive oxygen species (ROS) and deplete GSH to efficiently induce cell apoptosis under hypoxic conditions, which has a high inhibiting effect on the growth of tumors, offering an attractive strategy to amplify CO and ROS generation to meet therapeutic requirements in cancer treatment.

Microscopic Mechanism for Further NO Heterogeneous Reduction by Potassium-Doped Biochar: A DFT Study.

Biomass reburning is an efficient and low-cost way to control nitric oxide (NO), and the abundant potassium (K) element in biomass affects the heterogeneous reaction between NO and biochar. Due to the incomplete simulation of the NO heterogeneous reduction reaction pathway at the molecular level and the unclear catalytic effect of K element in biochar, further research is needed on the possible next reaction and the influencing mechanism of the element. After the products of the existing reaction pathways are referenced, two reasonably simplified biochar structural models are selected as the basic reactants to study the microscopic mechanism for further NO heterogeneous reduction on the biochar surface before and after doping with the K atom based on density functional theory. In studying the two further NO heterogeneous reduction reaction pathways, we find that the carbon monoxide (CO) molecule fragment protrudes from the surface of biochar models with the desorption of N2 at the TS4 transition state, and the two edge types of biochar product models obtained by simulation calculation are Klein edge and ac56 edge observed in the experiment. In studying the catalytic effect of potassium in biochar, we find that the presence of K increases the heat release of adsorption of NO molecules, reduces the energy barrier of the rate-determining step in the nitrogen (N2) generation and desorption process (by 50.88 and 69.97%), and hinders the CO molecule from desorbing from the biochar model surface. Thermodynamic and kinetic analyses also confirm its influence. The study proves that the heterogeneous reduction reaction of four NO molecules on the surface of biochar completes the whole reaction process and provides a basic theoretical basis for the emission of nitrogen oxides (NOx) during biomass reburning.

Structural dynamics of the heme pocket and intersubunit coupling in the dimeric hemoglobin from Scapharca inaequivalvis.

Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.

CO Oxidation Mechanism of Silver-Substituted Mo/Cu CO-Dehydrogenase - Analogies and Differences to the Native Enzyme.

The aerobic oxidation of carbon monoxide to carbon dioxide is catalysed by the Mo/Cu-containing CO-dehydrogenase enzyme in the soil bacterium Oligotropha carboxidovorans, enabling the organism to grow on the small gas molecule as carbon and energy source. It was shown experimentally that silver can be substituted for copper in the active site of Mo/Cu CODH to yield a functional enzyme. In this study, we employed QM/MM calculations to investigate whether the reaction mechanism of the silver-substituted enzyme is similar to that of the native enzyme. Our results suggest that the Ag-substituted enzyme can oxidize CO and release CO2 following the same reaction steps as the native enzyme, with a computed rate-limiting step of 10.4 kcal/mol, consistent with experimental findings. Surprisingly, lower activation energies for C-O bond formation have been found in the presence of silver. Furthermore, comparison of rate constants for reduction of copper- and silver-containing enzymes suggests a discrepancy in the transition state stabilization upon silver substitution. We also evaluated the effects that differences in the water-active site interaction may exert on the overall energy profile of catalysis. Finally, the formation of a thiocarbonate intermediate along the catalytic pathway was found to be energetically unfavorable for the Ag-substituted enzyme. This finding aligns with the hypothesis proposed for the wild-type form, suggesting that the creation of such species may not be necessary for the enzymatic catalysis of CO oxidation.