Histidine Imidazole Research Articles

A strategy for improving the mechanical properties of silk fibers through the combination of genetic manipulation and zinc ion crosslinking.

Silk fiber is generally considered an excellent biological material due to its good biocompatibility, morphological plasticity and biodegradability. Previously, the construction of silkworm silk gland bioreactors based on the piggyBac transposon has been optimized. However, the inserted exogenous genes have problems such as position uncertainty, and expression is not strictly controlled. Here, we applied transcription activator-like effector nuclease (TALEN)-mediated homology-directed repair (HDR) to precisely insert histidine-rich cuticular protein (CP) into silkworm Sericin1 (Ser1) gene. The Ser1-CP fusion protein was successfully secreted into cocoon shell. Subsequently, based on the metal coordination ability of the histidine imidazole group, we crosslinked cocoon with metal ions in vitro. In this strategy, the mechanical properties of the fused silk fibers with crosslinked Zn2+ improved, and the maximum breaking stress of the crosslinked Zn2+-fused silk fibers was 23.5 % greater than that of the wild-type fibers. Analysis of the secondary structure of the silk protein showed that the fused silk fibers crosslinked with Zn2+ had more β-sheet structures. This study pioneered a method of improving the mechanical properties of silk fibers by crosslinking metal ions with fused exogenous proteins and expanded the application value of silk gland bioreactors in the development of novel biomaterials.

Electron Paramagnetic Resonance Analysis of Hydroquinone Polymerization Catalyzed by Small Laccase

AbstractLaccases are multi‐copper oxidases (MCOs) that oxidize a broad range of substrates while reducing molecular oxygen to water. Although much effort has been made to elucidate the catalytic mechanism of laccases, information about reactive catalytic intermediates during catalysis is not yet fully understood. Herein, hydroquinone (HQ) polymerization catalyzed by prokaryotic small laccase (SLAC) was investigated by electron paramagnetic resonance (EPR) spectroscopy to provide more information on the catalytic mechanism. Briefly, free radical intermediates during the catalysis were investigated by real‐time in situ EPR measurement to monitor the formation and transformation of free radicals. A paramagnetic species was identified which was attributed to a copolymer formed by hydroquinone, benzoquinone, and semiquinone radical. In addition, the low‐temperature EPR spectrum of the trinuclear copper center during catalysis not only revealed tentative rotational motion of the histidine imidazole rings coordinating the TNC upon electron uptake but also provided evidence of the formation of oxygen bridge at TNC during the reaction, represented by the observation of a new type 2 copper component featured larger g// values and smaller A// values. Together, EPR spectroscopic insights into the catalytic intermediates during enzymatic hydroquinone polymerization have extended the knowledge of the biophysical characteristics and catalytic mechanism of SLAC.

Simulating Metal-Imidazole Complexes.

One commonly observed binding motif in metalloproteins involves the interaction between a metal ion and histidine's imidazole side chains. Although previous imidazole-M(II) parameters established the flexibility and reliability of the 12-6-4 Lennard-Jones (LJ)-type nonbonded model by simply tuning the ligating atom's polarizability, they have not been applied to multiple-imidazole complexes. To fill this gap, we systematically simulate multiple-imidazole complexes (ranging from one to six) for five metal ions (Co(II), Cu(II), Mn(II), Ni(II), and Zn(II)) which commonly appear in metalloproteins. Using extensive (40 ns per PMF window) sampling to assemble free energy association profiles (using OPC water and standard HID imidazole charge models from AMBER) and comparing the equilibrium distances to DFT calculations, a new set of parameters was developed to focus on energetic and geometric features of multiple-imidazole complexes. The obtained free energy profiles agree with the experimental binding free energy and DFT calculated distances. To validate our model, we show that we can close the thermodynamic cycle for metal-imidazole complexes with up to six imidazole molecules in the first solvation shell. Given the success in closing the thermodynamic cycles, we then used the same extended sampling method for six other metal ions (Ag(I), Ca(II), Cd(II), Cu(I), Fe(II), and Mg(II)) to obtain new parameters. Since these new parameters can reproduce the one-imidazole geometry and energy accurately, we hypothesize that they will reasonably predict the binding free energy of higher-level coordination numbers. Hence, we did not extend the analysis of these ions up to six imidazole complexes. Overall, the results shed light on metal-protein interactions by emphasizing the importance of ligand-ligand interaction and metal-π-stacking within metalloproteins.

N‐methylation of histidine to tune tautomeric preferences in histidine‐heme coordination and enzyme‐mimetic catalysis

AbstractEnzymes with active sites involving histidine selectively utilize either the δ‐ or ε‐nitrogen atom (Nδ or Nε) of the histidine imidazole for catalysis. However, evaluating the impact of Nδ and Nε is difficult, and directly integrating noncanonical N‐methylated histidine within enzymes poses risks due to laborious procedures. In this study, we present the self‐assembly of Fmoc‐Histidine (Fmoc‐His) with hemin to create a peroxidase‐mimetic catalyst, in which either the Nε or Nδ of histidine is methylated to modify the tautomeric preferences, thereby tuning hemin catalysis. UV‐vis spectra, 1H‐NMR, and fluorescence experiments elucidate that the N‐methylation of histidine alters the self‐assembly propensity of Fmoc‐His, and affects the binding affinity of histidine to hemin iron, with Fmoc‐δmHis/hemin exhibiting stronger binding than Fmoc‐εmHis/hemin. Theoretical simulation results suggest that εmHis and δmHis ligation produce a saddled structure and planar structure of hemin, respectively, stemming from the disparity of steric hindrance at the Nε and Nδ positions. The significant inhibition of hemin's oxidative activity by Fmoc‐δmHis is observed, likely due to the strong binding of Fmoc‐δmHis, potentially hindering access of the substrate, H2O2, to the hemin iron. Conversely, Fmoc‐εmHis enhances hemin catalysis, surpassing even Fmoc‐His alone. This differential impact of Fmoc‐εmHis and Fmoc‐δmHis on hemin activity is further corroborated by apparent activation energy and kinetic parameters (kcat, kcat/Km). This study sheds light on the heterogeneous biological effects at the nitrogen positions of histidine imidazole and offers insights into designing supramolecular metalloenzymes.

Iodide enhances degradation of histidine sidechain and imidazoles and forms new iodinated aromatic disinfection byproducts during chlorination

Peptide-bound histidines and imidazoles are important constituents of dissolved organic matter in water, and understanding the formation of halogenated disinfection byproduct (DBP) formation from these compounds during disinfection is important for ensuring a safe drinking water supply. Previous studies suggested that histidine has low reactivity with chlorine only; this study indicates that iodide substantially enhances histidine reactivity with the disinfectant at a time scale from days to hours. Mono- and di-iodinated histidines were identified as dominant transformation products with cumulative molar yields of 3.3 % at 6 h and they were stable in water over 7 days. These products were formed via electrophilic substitution of iodine to imidazole ring when hypoiodous acid reacted with histidine sidechain. Bromide minimally influenced the formation yields of these iodinated products, and higher pH increased yields up to 12 % for pH in the range 5–9. The cumulative concentration of low-molecular-weight DBPs, such as trihalomethanes and haloacetic acids, was less than 0.3 % under the same conditions. Similar iodinated imidazole analogs were also identified from other imidazoles (i.e., imidazole-carboxylic and phenyl-imidazole-carboxylic acids). This study demonstrated that peptide-bound histidine and imidazoles can serve as important precursors to iodinated aromatic DBPs, facilitating the identification of less-known iodinated DBPs.

Co(II) Substitution Enhances the Esterase Activity of a de Novo Designed Zn(II) Carbonic Anhydrase.

Carbonic Anhydrases (CAs) have been a target for de novo protein designers due to the simplicity of the active site and rapid rate of the reaction. The first reported mimic contained a Zn(II) bound to three histidine imidazole nitrogens and an exogenous water molecule, hence closely mimicking the native enzymes' first coordination sphere. Co(II) has served as an alternative metal to interrogate CAs due to its d7 electronic configuration for more detailed solution characterization. We present here the Co(II) substituted [Co(II)(H2O/OH-)]N(TRIL2WL23H)3 n+ that behaves similarly to native Co(II) substituted human-CAs. Like the Zn(II) analogue, the cobalt-derivative at slightly basic pH is incapable of hydrolyzing p-nitrophenylacetate (pNPA); however, as the pH is increased a significant activity develops, which at pH values above 10 eventually yields a catalytic efficiency that exceeds that of the [Zn(II)(OH-)]N(TRIL2WL23H)3 + peptide complex. X-ray absorption analysis is consistent with an octahedral species at pH 7.5 that converts to a 5-coordinate species by pH 11. UV-vis spectroscopy can monitor this transition, giving a pKa for the conversion of 10.3. We assign this conversion to the formation of a 5-coordinate Co(II)(Nimid)3(OH)(H2O) species. The pH dependent kinetic analysis indicates the maximal rate (kcat), and thus the catalytic efficiency (kcat/Km), follow the same pH profile as the spectroscopic conversion to the pentacoordinate species. This correlation suggests that the chemically irreversible ester hydrolysis corresponds to the rate determining process.

Significance of Histidine Hydrogen-Deuterium Exchange Mass Spectrometry in Protein Structural Biology.

Histidine residues play crucial roles in shaping the function and structure of proteins due to their unique ability to act as both acids and bases. In other words, they can serve as proton donors and acceptors at physiological pH. This exceptional property is attributed to the side-chain imidazole ring of histidine residues. Consequently, determining the acid-base dissociation constant (Ka) of histidine imidazole rings in proteins often yields valuable insights into protein functions. Significant efforts have been dedicated to measuring the pKa values of histidine residues in various proteins, with nuclear magnetic resonance (NMR) spectroscopy being the most commonly used technique. However, NMR-based methods encounter challenges in assigning signals to individual imidazole rings and require a substantial amount of proteins. To address these issues associated with NMR-based approaches, a mass-spectrometry-based method known as histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) has been developed. This technique not only determines the pKa values of histidine imidazole groups but also quantifies their solvent accessibility. His-HDX-MS has proven effective across diverse proteins, showcasing its utility. This review aims to clarify the fundamental principles of His-HDX-MS, detail the experimental workflow, explain data analysis procedures and provide guidance for interpreting the obtained results.

Lasso Peptides: Exploring the Folding Landscape of Nature's Smallest Interlocked Motifs.

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

Structural features of the active centre and quaternary structure of inulinase

Now many researchers started to pay attention to the fact that not only traditional sorbents and ion exchangers, but also a number of natural components contained in plant and animal organisms have the ability to absorb other organic molecules and ions, enter into complex formation reactions with metal ions, and exhibit catalytic properties. Such natural substances with sorption and ion-exchange properties include aminopolysaccharides (chitin, chitosan), nucleic and ribonucleic acids, saponins, and enzymes. The provided list of substances can be classified as high-molecular compounds with sorption, ligand and catalytic properties. This study presents the results of the investigation of the functional groups of the active centre of inulinase and the features of its quaternary structure. It was established that the enzyme has a quaternary structure, represented by two subunits with Mr 76900 Da and 10140 Da, which have catalytic activity. The imidazole radical of histidine, SH groups and carboxyl groups take part in the formation of the enzyme-substrate complex. The purpose of this study was investigation of the functional groups of the active centre and some features of the quaternary structure of inulinase. In this study, we used a preparation of inulinase isolated from Aspergillus awamori Ts2250, purified by ion exchange chromatography on columns with diethylaminoethylcellulose (DEAE): [-O-(CH2)2-N(C2H5)2]. The homogeneity of the drug was confirmed by gel electrophoresis [24-25]. The catalytic activity of inulinase was determined on the substrate inulin (Spofa, Czech Republic) spectrophotometrically using resorcinol at λ = 540 nm, and the molecular weight was determined by gel chromatography on Sephadex G-200. The presence of electrophilic groups COO- was determined using the Dixon method [28], and also by IR spectroscopy on a Vertex-70 device (Bruker, Germany) in the frequency range 4000-400 cm-1. The active centre of the enzyme includes γ and δ-carboxyl groups of aspartic and glutamic acid residues, respectively. Hydrogen ion H+ splits off from the carboxyl group of the glutamine residue of the enzyme and binds to oxygen connecting rings A and B of the substrate. As a result, the oxygen bond with the ring is broken, and the carbon located in position I of ring A forms a carbonium ion, which is stabilized by COO- group of the aspartic acid residue of the enzyme. An ОН- ion delivered by a water molecule interacts with carbonium ion, and H+ ion of water is fixed in the place of H+ ion lost by the glutamic acid residue in inulinase. After this, inulin molecules leave the enzyme, freeing it for subsequent reaction with the substrate. However, this mechanism is not the only one. Histidine imidazole also takes part in the formation of the enzyme-substrate complex. When interacting with inulin, the imidazole group is hydrogen bonded to the oxygen connecting rings A and B of the substrate. There is also an orientation of the COO- ion of the enzyme ion relative to the resulting carbonium ion in ring A. Subsequently, Н+ and ОН- ions from water molecules are fixed in inulinase in place of H+, lost Glu, and OH- is fixed on the carbonium ion of inulin.

A Cobalt(III) Corrole with a Tethered Imidazole for Boosted Electrocatalytic Oxygen Reduction Reaction

Comprehensive SummaryDeveloping electrocatalysts with high activity and selectivity for the oxygen reduction reaction (ORR) is vital to promote the performance of the next‐generation energy technologies, which depend on the efficiency of the catalytic reduction of dioxygen. In the structure of cytochrome c oxidases (CcOs), a histidine imidazole residue binding to the axial position of Fe plays a crucial role in facilitating the selective reduction of O2 to water. Inspired by nature, we herein report on the synthesis of CoIII corrole 1 tethered with an imidazole ligand as well as its electrocatalytic ORR and O2 binding features. As compared to the imidazolium‐free analogue, complex 1 displayed remarkably boosted activity for the selective four‐electron/four‐proton (4e‐/4H+) ORR with a half‐wave potential of E1/2 = 0.82 V versus reversible hydrogen electrode (RHE) in 0.1 mol/L KOH solutions. Importantly, we demonstrate that the tethered axial imidazole ligand improves the O2 binding ability of 1 thermodynamically and dynamically, which is crucial to boost electrocatalytic ORR performance. This work presents an example to improve electrocatalytic ORR activity and selectivity of Co corroles by introducing an axial imidazole ligand to enhance the O2 binding and activation.

Structural correlations of complexation of apple pectin with imidazole and L-histidine methyl ester

The thermodynamic and structural study of the complex formation of apple pectin with structural analogs of histidine (imidazole and its methyl ester) was carried out using spectral methods. The composition, stability constants of the complexes, and standard thermodynamic characteristics (Δ G° , Δ H° , Δ S° ) of the complex formation process were determined. The decisive contribution of the imidazole fragment of the amino acid to the stability of the pectin-histidine complex is shown. The esterification of the carboxyl group of histidine, its conversion into methyl ester, has an insignificant effect on the efficiency of complex formation with pectin, leading only to a slight increase in binding.

Films, Gels and Electrospun Fibers from Serum Albumin Globular Protein for Medical Device Coating, Biomolecule Delivery and Regenerative Engineering.

Albumin is a natural biomaterial that is abundantly available in blood and body fluids. It is clinically used as a plasma expander, thereby increasing the plasma thiol concentration due to its cysteine residues. Albumin is a regulator of intervascular oncotic pressure, serves as an anti-inflammatory modulator, and it has a buffering role due to its histidine imidazole residues. Because of its unique biological and physical properties, albumin has also emerged as a suitable biomaterial for coating implantable devices, for cell and drug delivery, and as a scaffold for tissue engineering and regenerative medicine. As a biomaterial, albumin can be used as surface-modifying film or processed either as cross-linked protein gels or as electrospun fibers. Herein we have discussed how albumin protein can be utilized in regenerative medicine as a hydrogel and as a fibrous mat for a diverse role in successfully delivering drugs, genes, and cells to targeted tissues and organs. The review of prior studies indicated that albumin is a tunable biomaterial from which different types of scaffolds with mechanical properties adjustable for various biomedical applications can be fabricated. Based on the progress made to date, we concluded that albumin-based device coatings, delivery of drugs, genes, and cells are promising strategies in regenerative and personalized medicine.

PH Dependence of Amyloid-β Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes.

Central to Alzheimer's disease (AD) is the assembly of the amyloid-beta peptide (Aβ) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aβ's isoelectric point (pI). Using global fitting of fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual fibril assembly microscopic rate constants. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid-point or pK a of 7.0, close to the pK a of Aβ histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aβ, in particular histidine protonation, has little impact on this stage of Aβ assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aβ into toxic oligomers and fibrils.

PH Dependence of Amyloid-β Fibril Assembly Kinetics: Unravelling the Microscopic Molecular Processes.

Central to Alzheimer's disease (AD) is the assembly of the amyloid-beta peptide (Aβ) into fibrils. A reduction in pH accompanying inflammation or subcellular compartments, may accelerate fibril formation as the pH approaches Aβ's isoelectric point (pI). Using global fitting of fibril formation kinetics over a range of pHs, we identify the impact net charge has on individual fibril assembly microscopic rate constants. We show that the primary nucleation has a strong pH dependence. The titration behaviour exhibits a mid-point or pKa of 7.0, close to the pKa of Aβ histidine imidazoles. Surprisingly, both the secondary nucleation and elongation rate constants are pH independent. This indicates the charge of Aβ, in particular histidine protonation, has little impact on this stage of Aβ assembly. These fundamental processes are key to understanding the forces that drive the assembly of Aβ into toxic oligomers and fibrils.

The Nϵ-Rule for Serine, but Not Cysteine Catalytic Triads.

Catalytic triads, composed of a serine or cysteine nucleophile, a histidine, and a third triad residue (typically Asp/Glu/Asn), are common in enzyme active sites and catalyze a wide variety of chemical reactions. Two types of triads can be distinguished: We refer to them as Nδ- or Nϵ-configured, depending on whether the histidine imidazole Nδ or Nϵ atom is close to the nucleophile Oγ/Sγ. In this study, we have analyzed triad configuration. In structural triads, the more stable Nδ-configuration predominates. For catalytic triads, the configuration depends on the nucleophile. When it is a cysteine residue, both configuration types occur, depending on the family. However, when the nucleophile is a serine residue, the less stable Nϵ-configuration is almost exclusively found. We posit that the energetically less favored conformation is selected for in serine triads to facilitate the otherwise difficult proton transfer from the nucleophile to the histidine residue.

The Nϵ‐Rule for Serine, but Not Cysteine Catalytic Triads

Catalytic triads, composed of a serine or cysteine nucleophile, a histidine, and a third triad residue (typically Asp/Glu/Asn), are common in enzyme active sites and catalyze a wide variety of chemical reactions. Two types of triads can be distinguished: We refer to them as Nδ- or Nϵ-configured, depending on whether the histidine imidazole Nδ or Nϵ atom is close to the nucleophile Oγ/Sγ. In this study, we have analyzed triad configuration. In structural triads, the more stable Nδ-configuration predominates. For catalytic triads, the configuration depends on the nucleophile. When it is a cysteine residue, both configuration types occur, depending on the family. However, when the nucleophile is a serine residue, the less stable Nϵ-configuration is almost exclusively found. We posit that the energetically less favored conformation is selected for in serine triads to facilitate the otherwise difficult proton transfer from the nucleophile to the histidine residue.

Thermodynamic surprises of Cu(II)\u2013amylin analogue complexes in membrane mimicking solutions

Membrane environment often has an important effect on the structure, and therefore also on the coordination mode of biologically relevant metal ions. This is also true in the case of Cu(II) coordination to amylin analogues—rat amylin, amylin1–19, pramlintide and Ac-pramlintide, which offer N-terminal amine groups and/or histidine imidazoles as copper(II) anchoring sites. Complex stabilities are comparable, with the exception of the very stable Cu(II)–amylin1–19, which proves that the presence of the amylin C-terminus lowers its affinity for copper(II); although not directly involved, its appropriate arrangement sterically prevents early metal binding. Most interestingly, in membrane-mimicking solution, the Cu(II) affinities of amylin analogues are lower than the ones in water, probably due to the crowding effect of the membrane solution and the fact that amide coordination occurs at higher pH, which happens most likely because the α-helical structure, imposed by the membrane-mimicking solvent, prevents the amides from binding at lower pH, requiring a local unwinding of the α-helix.

Modified histidine containing amphipathic ultrashort antifungal peptide, His[2-p-(n-butyl)phenyl]-Trp-Arg-OMe exhibits potent anticryptococcal activity.

In pursuit of ultrashort peptide-based antifungals, a new structural class, His(2-aryl)-Trp-Arg is reported. Structural changes were investigated on His-Trp-Arg scaffold to demonstrate the impact of charge and lipophilic character on the biological activity. The presence and size of the aryl moiety on imidazole of histidine modulated overall amphiphilic character, and biological activity. Peptides exhibited IC50 of 0.37-9.66μg/mL against C.neoformans. Peptide 14f [His(2-p-(n-butyl)phenyl)-Trp-Arg-OMe] exhibited two-fold potency (IC50=0.37μg/mL, MIC=0.63μg/mL) related to amphotericin B, without any cytotoxic effects up to 10μg/mL. Peptide 14f act by nuclear fragmentation, membranes permeabilization, disruption and pore formations in the microbial cells as determined by the mechanistic studies employing Trp-quenching, CLSM, SEM, and HR-TEM. The amalgamation of short sequence, presence of appropriate aryl group on l-histidine, potent anticryptococcal activity, no cytotoxicity, and detailed mechanistic studies directed to the identification of 14f as a new antifungal structural lead.

Binding of histidine and human serum albumin to dirhodium(II) tetraacetate

Reactions of the anticancer active dirhodium tetraacetate (1), Rh2(AcO)4 (AcO− = CH3COO−), with the amino acid histidine (HHis) and human serum albumin (HSA) were monitored over time and different metal: ligand ratios using UV-vis spectroscopy and/or electro-spray ionization mass spectrometry. Initially, histidine formed 1:1 and 1:2 adducts in aqueous solutions. The crystal structure of Rh2(AcO)4(L-HHis)2·2H2O (2) confirmed the axial coordination of histidine imidazole groups (average Rh-Naxial 2.23 Å). These adducts, however, were found to be unstable in solution over time (24 h). Heating Rh2(AcO)4 –histidine solutions to 40 °C (near body temperature) or 95 °C accelerated the formation of RhII2(AcO)2(His)2 and RhIII(His)2(AcO) complexes. The corresponding pH change from neutral to mildly acid (pH 4–5) indicates deprotonation of histidine NH3+ groups due to coordination to Rh ions, which simultaneously bind to histidine COO− groups, as evidenced by 13C NMR spectroscopy. In the case of HSA with 16 histidine and one cysteine residues, UV-vis spectroscopy indicates that mono- and di-histidine HSA adducts with Rh2(AcO)4 are formed. X-ray absorption spectroscopy showed almost the same Rh-Rh distance (2.41 ± 0.01 Å) for the Rh2(AcO)4 units as in 2, and a contribution from an axial thiol coordination (Rh-Saxial 2.62 ± 0.05 Å). The Rh2(AcO)4 – HSA complex was found to decompose partially (~15%) over 24 h at ambient temperature. The partial decomposition of Rh2(AcO)4 both through coordination to histidine or to human serum albumin, the most abundant protein in blood plasma, is a factor to consider for its efficacy as a potential anticancer agent.

Two-dimensional HYSCORE spectroscopy reveals a histidine imidazole as the axial ligand to Chl3A in the M688HPsaA genetic variant of Photosystem I

Recent studies on Photosystem I (PS I) have shown that the six core chlorophyll a molecules are highly coupled, allowing for efficient creation and stabilization of the charge-separated state. One area of particular interest is the identity and function of the primary acceptor, A0, as the factors that influence its ultrafast processes and redox properties are not yet fully elucidated. It was recently shown that A0 exists as a dimer of the closely-spaced Chl2/Chl3 molecules wherein the reduced A0− state has an asymmetric distribution of electron spin density that favors Chl3. Previous experimental work in which this ligand was changed to a hard base (histidine, M688HPsaA) revealed severely impacted electron transfer processes at both the A0 and A1 acceptors; molecular dynamics simulations further suggested two distinct conformations of PS I in which the His residue coordinates and forms a hydrogen bond to the A0 and A1 cofactors, respectively. In this study, we have applied 2D HYSCORE spectroscopy in conjunction with molecular dynamics simulations and density functional theory calculations to the study of the M688HPsaA variant. Analysis of the hyperfine parameters demonstrates that the His imidazole serves as the axial ligand to the central Mg2+ ion in Chl3A in the M688HPsaA variant. Although the change in ligand identity does not alter delocalization of electron density over the Chl2/Chl3 dimer, a small shift in the asymmetry of delocalization, coupled with the electron withdrawing properties of the ligand, most likely accounts for the inhibition of forward electron transfer in the His-ligated conformation.