Subtle distinction in molecular structure of flavonoids leads to vastly different coating efficiency and mechanism of metal-polyphenol networks with excellent antioxidant activities.

Metal-polyphenol networks (MPNs) are of immense scientific interest because of their simple and rapid process to deposit on various substrates or particles with different shapes. However, there are rare reports on the effect of polyphenol molecular structure on coating efficiency and mechanism of MPNs. Herein, three typical flavonoid polyphenols, catechin (Cat), epigallocatechin (EGC) and procyanidin (PC), with the same skeleton (C6-C3-C6) but subtle distinction in molecular structure, were selected to build MPN coatings with ferric ions (Fe3+). And various techniques combined with the density functional theory (DFT) were applied to deeply reveal the roles of coordinative phenolic hydroxyl groups as well as noncovalent interactions (hydrogen bonding and π - π stacking) in the formation of flavonoid-based MPNs. We found that more accessible numbers of coordinative phenolic hydroxyl groups, the higher coating efficiency. In these flavonoid-based MPNs, the single-complex is the predominant during the coordinative modes between phenolic hydroxyl and Fe3+, not the previously reported mono-complex, bis-complex and/or tris-complex. Besides coordinative interaction, noncovalent interactions also contribute to MPNs formation, and hydrogen bonds prevail in the noncovalent interaction compared with π-π stacking. And these engineered MPN coatings can endow the substrate with excellent antioxidant activities. This study contributes to in-depth understanding the building mechanism of flavonoid-based MPNs, and increasing coating efficiency by choosing proper polyphenols.

Mechanistic Origin of Favorable Substituent Effects in Excellent Cu Cyclam Based HNO Sensors.

HNO has broad chemical and biomedical properties. Metal complexes and derivatives are widely used to make excellent HNO sensors. However, their favorable mechanistic origins are largely unknown. Cu cyclam is a useful platform to make excellent HNO sensors including imaging agents. A quantum chemical study of Cu cyclams with various substitutions was performed, which reproduced diverse experimental reactivities. Structural, electronic, and energetic profiles along reaction pathways show the importance of HNO binding and a proton-coupled electron transfer mechanism for HNO reaction. Results reveal that steric effect is primary and electronic factor is secondary (if the redox potential is sufficient), but their interwoven effects can lead to unexpected reactivity, which looks mysterious experimentally but can be explained computationally. This work suggests rational substituent design ideas and recommends a theoretical study of a new design to save time and cost due to its subtle effect.

HNO to NO Conversion Mechanism with Copper Zinc Superoxide Dismutase, Comparison with Heme Protein Mediated Conversions, and the Origin of Questionable Reversibility.

The interconversion of NO and HNO, via copper zinc superoxide dismutase (CuZnSOD), is important in biomedicine and for HNO detection. Many mechanistic questions, including the decades-long debate on reversibility, were resolved in this work. Calculations of various active-site and full-protein models show that the basic mechanism is proton-coupled electron transfer with a computed barrier of 10.98 kcal mol-1 , which is in excellent agreement with experimental results (10.62 kcal mol-1 ), and this nonheme protein-mediated reaction has many significant mechanistic differences compared with the conversions mediated by heme proteins due to geometric and electronic factors. The reasons for the irreversible nature of this conversion and models with the first thermodynamically favorable and kinetically feasible mechanism for the experimental reverse reaction were discovered. Such results are the first for nonheme enzyme mediated HNO to NO conversions, which shall facilitate other related studies and HNO probe development.

The oxygen reactivity of an artificial hydrogenase designed in a reengineered copper storage protein.

The O2 reactivity of an artificial biomolecular hydrogenase, the nickel binding protein (NBP) is investigated. Kinetic analyses revealed a complete 4e- reduction of O2 to H2O under catalytic conditions with associated k0 for ET in the order of 10-6 cm s-1. Protein destabilization and S oxygenation are contributing factors to the deactivation of NBP under oxic conditions. Computational studies provided insight into the S oxygenation and the reaction intermediates of a proposed mechanistic pathway for O2 activation by NBP.

Redesign of a Copper Storage Protein into an Artificial Hydrogenase.

We report the construction of an artificial hydrogenase (ArH) by reengineering a Cu storage protein (Cspl) into a Ni-binding protein (NBP) employing rational metalloprotein design. The hypothesis driven design approach involved deleting existing Cu sites of Csp1 and identification of a target tetrathiolate Ni binding site within the protein scaffold followed by repacking the hydrophobic core. Guided by modeling, the NBP was expressed and purified in high purity. NBP is a well-folded and stable construct displaying native-like unfolding behavior. Spectroscopic and computational studies indicated that the NBP bound nickel in a distorted square planar geometry that validated the design. Ni(II)-NBP is active for photo-induced H2 evolution following a reductive quenching mechanism. Ni(II)-NBP catalyzed H+ reduction to H2 gas electrochemically as well. Analysis of the catalytic voltammograms established a proton-coupled electron transfer (PCET) mechanism. Electrolysis studies confirmed H2 evolution with quantitative Faradaic yields. Our studies demonstrate an important scope of rational metalloprotein design that allows imparting functions into protein scaffolds that have natively not evolved to possess the same function of the target metalloprotein constructs.

Mechanisms of HNO Reactions with Ferric Heme Proteins.

Many HNO-scavenging pathways exist to regulate its biological and pharmacological activities. Such reactions often involve ferric heme proteins and form an important basis for HNO probe development. However, mechanisms of HNO reactions with ferric heme proteins are largely unknown. We performed a computational investigation using metmyoglobin and catalase as representative ferric heme proteins with neutral and negatively charged axial ligands to provide the first detailed pathways. The results reproduced experimental barriers well with an average error of 0.11 kcal mol-1 . The rate-limiting step was found to be dissociation of the resting ligand or HNO coordination when there is no resting ligand. For both heme proteins, in contrast to the non-heme case, the reductive nitrosylation step was found to be barrierless proton-coupled electron transfer, which provides the major thermodynamic driving force for the overall reaction. The origin of the difference in reactivity between metmyoglobin and catalase was also revealed.

Nitrosyl Myoglobins and Their Nitrite Precursors: Crystal Structural and Quantum Mechanics and Molecular Mechanics Theoretical Investigations of Preferred Fe -NO Ligand Orientations in Myoglobin Distal Pockets.

The globular dioxygen binding heme protein myoglobin (Mb) is present in several species. Its interactions with the simple nitrogen oxides, namely, nitric oxide (NO) and nitrite, have been known for decades, but the physiological relevance has only recently become more fully appreciated. We previously reported the O-nitrito mode of binding of nitrite to ferric horse heart wild-type (wt) MbIII and human hemoglobin. We have expanded on this work and report the interactions of nitrite with wt sperm whale (sw) MbIII and its H64A, H64Q, and V68A/I107Y mutants whose dissociation constants increase in the following order: H64Q < wt < V68A/I107Y < H64A. We also report their X-ray crystal structures that reveal the O-nitrito mode of binding of nitrite to these derivatives. The MbII-mediated reductions of nitrite to NO and structural data for the wt and mutant MbII-NOs are described. We show that their FeNO orientations vary with distal pocket identity, with the FeNO moieties pointing toward the hydrophobic interiors when the His64 residue is present but toward the hydrophilic exterior when this His64 residue is absent in this set of mutants. This correlates with the nature of H-bonding to the bound NO ligand (nitrosyl O vs N atom). Quantum mechanics and hybrid quantum mechanics and molecular mechanics calculations help elucidate the origin of the experimentally preferred NO orientations. In a few cases, the calculations reproduce the experimentally observed orientations only when the whole protein is taken into consideration.

Heme redox potentials hold the key to reactivity differences between nitric oxide reductase and heme-copper oxidase.

Despite high structural homology between NO reductases (NORs) and heme-copper oxidases (HCOs), factors governing their reaction specificity remain to be understood. Using a myoglobin-based model of NOR (FeBMb) and tuning its heme redox potentials (E°') to cover the native NOR range, through manipulating hydrogen bonding to the proximal histidine ligand and replacing heme b with monoformyl (MF-) or diformyl (DF-) hemes, we herein demonstrate that the E°' holds the key to reactivity differences between NOR and HCO. Detailed electrochemical, kinetic, and vibrational spectroscopic studies, in tandem with density functional theory calculations, demonstrate a strong influence of heme E°' on NO reduction. Decreasing E°' from +148 to -130 mV significantly impacts electronic properties of the NOR mimics, resulting in 180- and 633-fold enhancements in NO association and heme-nitrosyl decay rates, respectively. Our results indicate that NORs exhibit finely tuned E°' that maximizes their enzymatic efficiency and helps achieve a balance between opposite factors: fast NO binding and decay of dinitrosyl species facilitated by low E°' and fast electron transfer facilitated by high E°'. Only when E°' is optimally tuned in FeBMb(MF-heme) for NO binding, heme-nitrosyl decay, and electron transfer does the protein achieve multiple (>35) turnovers, previously not achieved by synthetic or enzyme-based NOR models. This also explains a long-standing question in bioenergetics of selective cross-reactivity in HCOs. Only HCOs with heme E°' in a similar range as NORs (between -59 and 200 mV) exhibit NOR reactivity. Thus, our work demonstrates efficient tuning of E°' in various metalloproteins for their optimal functionality.

Manganese and Cobalt in the Nonheme-Metal-Binding Site of a Biosynthetic Model of Heme-Copper Oxidase Superfamily Confer Oxidase Activity through Redox-Inactive Mechanism.

The presence of a nonheme metal, such as copper and iron, in the heme-copper oxidase (HCO) superfamily is critical to the enzymatic activity of reducing O2 to H2O, but the exact mechanism the nonheme metal ion uses to confer and fine-tune the activity remains to be understood. We herein report that manganese and cobalt can bind to the same nonheme site and confer HCO activity in a heme-nonheme biosynthetic model in myoglobin. While the initial rates of O2 reduction by the Mn, Fe, and Co derivatives are similar, the percentages of reactive oxygen species (ROS) formation are 7%, 4%, and 1% and the total turnovers are 5.1 ± 1.1, 13.4 ± 0.7, and 82.5 ± 2.5, respectively. These results correlate with the trends of nonheme-metal-binding dissociation constants (35, 22, and 9 µM) closely, suggesting that tighter metal binding can prevent ROS release from the active site, lessen damage to the protein, and produce higher total turnover numbers. Detailed spectroscopic, electrochemical, and computational studies found no evidence of redox cycling of manganese or cobalt in the enzymatic reactions and suggest that structural and electronic effects related to the presence of different nonheme metals lead to the observed differences in reactivity. This study of the roles of nonheme metal ions beyond the Cu and Fe found in native enzymes has provided deeper insights into nature's choice of metal ion and reaction mechanism and allows for finer control of the enzymatic activity, which is a basis for the design of efficient catalysts for the oxygen reduction reaction in fuel cells.

Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin-based Functional Oxidase.

Heme-copper oxidase (HCO) is a class of respiratory enzymes that use a heme-copper center to catalyze O2 reduction to H2 O. While heme reduction potential (E°') of different HCO types has been found to vary >500 mV, its impact on HCO activity remains poorly understood. Here, we use a set of myoglobin-based functional HCO models to investigate the mechanism by which heme E°' modulates oxidase activity. Rapid stopped-flow kinetic measurements show that increasing heme E°' by ca. 210 mV results in increases in electron transfer (ET) rates by 30-fold, rate of O2 binding by 12-fold, O2 dissociation by 35-fold, while decreasing O2 affinity by 3-fold. Theoretical calculations reveal that E°' modulation has significant implications on electronic charge of both heme iron and O2 , resulting in increased O2 dissociation and reduced O2 affinity at high E°' values. Overall, this work suggests that fine-tuning E°' in HCOs and other heme enzymes can modulate their substrate affinity, ET rate and enzymatic activity.

HNO-Binding in Heme Proteins: Effects of Iron Oxidation State, Axial Ligand, and Protein Environment.

HNO plays significant roles in many biological processes. Numerous heme proteins bind HNO, an important step for its biological functions. A systematic computational study was performed to provide the first detailed trends and origins of the effects of iron oxidation state, axial ligand, and protein environment on HNO binding. The results show that HNO binds much weaker with ferric porphyrins than corresponding ferrous systems, offering strong thermodynamic driving force for experimentally observed reductive nitrosylation. The axial ligand was found to influence HNO binding through its trans effect and charge donation effect. The protein environment significantly affects the HNO hydrogen bonding structures and properties. The predicted NMR and vibrational data are in excellent agreement with experiment. This broad range of results shall facilitate studies of HNO binding in many heme proteins, models, and related metalloproteins.

HNO/NO Conversion Mechanisms of Cu-Based HNO Probes with Implications for Cu,Zn-SOD.

HNO has broad biological effects and pharmacological activities. Direct HNO probes for in vivo applications were recently reported, which are CuII-based complexes having fluorescence reporters with reaction to HNO resulting in CuI systems and the release of NO. Their coordination environments are similar to that in Cu,Zn-superoxide dismutase (SOD), which plays a significant role in cellular HNO/NO conversion. However, none of these conversion mechanisms are known. A quantum chemical investigation was performed here to provide structural, energetic, and electronic profiles of HNO/NO conversion pathways via the first CuII-based direct HNO probe. Results not only are consistent with experimental observations but also provide numerous structural and mechanistic details unknown before. Results also suggest the first HNO/NO conversion mechanism for Cu,Zn-SOD, as well as useful guidelines for future design of metal-based HNO probes. These results shall facilitate development of direct HNO probes and studies of HNO/NO conversions via metal complexes and metalloproteins.