Starch spectra of Ampelopteris prolifera (Retz.) Copel, a new addition to the existing lexicon and its comparison with a local potato cultivar (Solanum tuberosum L. cv. Kufri Jyoti).

Novel kinds of starch spectra were generated from a lesser-known plant, making this investigation unique. The recent trend of starch characterization shows the establishment of novel bioresources from nonconventional unexplored databases. The present endeavor was made to obtain the starch fingerprint of Ampelopteris prolifera (rhizome) belonging to seedless vascular plants. For comparison, a commercial local cultivar of potato (Kufri Jyoti) was taken. The starch particle of A. prolifera shows much uniqueness depicting its novelty viz., crystallinity index of 60.04 %, powder diffractogram at (2θ scale)17.57° to 39.78°; this diffractogram pattern is reported from this study as newer one i.e. R type(whereas potato starch is CB type); characteristic peak at 2θ = 20.07° suggests starch-lipid complex formation and V type crystallinity (i.e. RS 5 type); FTIR spectra showing the presence of more short chain branching; high gelatinization temperature(84.62 ±â€¯0.10), particle size and zeta value of A. prolifera is 4.00 ±â€¯0.81 µm and - 18.91 ±â€¯3.58 mV respectively. Bragg's peak from the single crystal X-ray diffraction has been generated for the first time of A. prolifera. Extraction of the starch particle was performed in chilled water. Therefore, the present study suggests wide-spectrum commercial utility and cost-effective production.

Recent Advances in the Phytochemistry of Bryophytes: Distribution, Structures and Biological Activity of Bibenzyl and Bisbibenzyl Compounds.

Research on bryophyte phytochemistry has revealed the presence of different phytochemicals like fatty acids, terpenoids, small phenolic molecules, etc. Small phenolic molecules, i.e., bibenzyls (of two aromatic rings) and bisbibenzyls (four aromatic rings), are unique signature molecules of liverworts. The first bisbibenzyls marchantin A and riccardin A were discovered in two consecutive years, i.e., 1982 and 1983, respectively, by Asakawa and coworkers. Since then, about 70 bisbibenzyls have been reported. These molecules are characterized and identified using different spectroscopic techniques and surveyed for different bioactivity and structure-activity relations. Biochemistry is determined by the season, geography, and environment. In this review, quantitative and qualitative information on bibenzyls and bisbibenzyl compounds and their distribution in different liverworts across, geographies along withtraditional to advanced extraction methods, and characterization techniques are summarized. Also, a comprehensive account of characteristic spectra of different bisbibenzyl compounds, their subtypes, and their basic skeleton patterns are compared. A comprehensive table is provided here for the first time presenting the quantity of bibenzyls, bisbenzyls, and their derivatives found in bryophytes, mentioning the spectroscopic data and mass profiles of the compounds. The significance of these compounds in different bioactivities like antibiotic, antioxidative, antitumor, antivenomous, anti-influenza, insect antifeedant, cytotoxic, and anticancerous activities are surveyed and critically enumerated.

Computational infrared and Raman spectra by hybrid QM/MM techniques: a study on molecular and catalytic material systems.

Vibrational spectroscopy is one of the most well-established and important techniques for characterizing chemical systems. To aid the interpretation of experimental infrared and Raman spectra, we report on recent theoretical developments in the ChemShell computational chemistry environment for modelling vibrational signatures. The hybrid quantum mechanical and molecular mechanical approach is employed, using density functional theory for the electronic structure calculations and classical forcefields for the environment. Computational vibrational intensities at chemical active sites are reported using electrostatic and fully polarizable embedding environments to achieve more realistic vibrational signatures for materials and molecular systems, including solvated molecules, proteins, zeolites and metal oxide surfaces, providing useful insight into the effect of the chemical environment on the signatures obtained from experiment. This work has been enabled by the efficient task-farming parallelism implemented in ChemShell for high-performance computing platforms.  This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

Multiscale QM/MM modelling of catalytic systems with ChemShell.

Hybrid quantum mechanical/molecular mechanical (QM/MM) methods are a powerful computational tool for the investigation of all forms of catalysis, as they allow for an accurate description of reactions occurring at catalytic sites in the context of a complicated electrostatic environment. The scriptable computational chemistry environment ChemShell is a leading software package for QM/MM calculations, providing a flexible, high performance framework for modelling both biomolecular and materials catalysis. We present an overview of recent applications of ChemShell to problems in catalysis and review new functionality introduced into the redeveloped Python-based version of ChemShell to support catalytic modelling. These include a fully guided workflow for biomolecular QM/MM modelling, starting from an experimental structure, a periodic QM/MM embedding scheme to support modelling of metallic materials, and a comprehensive set of tutorials for biomolecular and materials modelling.

Structures and Dynamics of Anionic Lipoprotein Nanodiscs.

Nanolipoprotein particles known as nanodiscs (NDs) have emerged as versatile and powerful tools for the stabilization of membrane proteins permitting a plethora of structural and biophysical studies. Part of their allure is their flexibility to accommodate many types of lipids and precise control of the composition. However, little is known about how variations in lipid composition impact their structures and dynamics. Herein, we investigate how the introduction of the anionic lipid POPG into POPC NDs impacts these features. Small-angle X-ray and neutron scattering (SAXS and SANS) of variable-composition NDs are complemented with molecular dynamics simulations to interrogate how increasing the concern of POPG impacts the ND shape, structure of the lipid core, and the dynamics of the popular membrane scaffold protein, MSP1D1(-). A convenient benefit of including POPG is that it eliminates D2O-induced aggregation observed in pure POPC NDs, permitting studies by SANS at multiple contrasts. SAXS and SANS data could be globally fit to a stacked elliptical cylinder model as well as an extension of the model that accounts for membrane curvature. Fitting to both models supports that the introduction of POPG results in strongly elliptical NDs; however, MD simulations predict the curvature of the membrane, thereby supporting the use of the latter model. Trends in the model-independent parameters suggest that increases in POPG reduce the conformational heterogeneity of the MSP1D1(-), which is in agreement with MD simulations that show that the incorporation of sufficient POPG suppresses disengagement of the N-terminal helix from the lipid core. These studies highlight novel structural changes in NDs in response to an anionic lipid and will inform the interpretation of future structural studies of membrane proteins embedded in NDs of mixed lipid composition.

QM/MM Simulations of Protein Crystal Reactivity Guided by MSOX Crystallography: A Copper Nitrite Reductase Case Study.

The recently developed multiple structures from one crystal (MSOX) serial crystallography method can be used to provide multiple snapshots of the progress of enzymatic reactions taking place within a protein crystal. Such MSOX snapshots can be used as a reference for combined quantum mechanical/molecular mechanical (QM/MM) simulations of enzyme reactivity within the crystal. QM/MM calculations are used to identify details of reference states that cannot be directly observed by X-ray diffraction experiments, such as protonation and oxidation states. These reference states are then used as known fixed endpoints for the modeling of reaction paths. We investigate the mechanism of nitrite reduction in an Achromobacter cycloclastes copper nitrite reductase crystal using MSOX-guided QM/MM calculations, identifying the change in nitrite binding orientation with a change in copper oxidation state, and determining the reaction path to the final NO-bound MSOX structure. The results are compared with QM/MM simulations performed in a solvated environment.

Reactive Molecular Dynamics at Constant Pressure via Nonreactive Force Fields: Extending the Empirical Valence Bond Method to the Isothermal-Isobaric Ensemble.

The Empirical Valence Bond (EVB) method offers a suitable framework to obtain reactive potentials through the coupling of nonreactive force fields. In this formalism, most of the implemented coupling terms are built using functional forms that depend on spatial coordinates, while parameters are fitted against reference data to model the change of chemistry between the participating nonreactive states. In this work, we demonstrate that the use of such coupling terms precludes the computation of the stress tensor for condensed phase systems and prevents the possibility to carry out EVB molecular dynamics in the isothermal-isobaric (NPT) ensemble. Alternatively, we make use of coupling terms that depend on the energy gaps, defined as the energy differences between the participating nonreactive force fields, and derive a general expression for the EVB stress tensor suitable for computation. Implementation of this new methodology is tested for a model of a single reactive malonaldehyde solvated in nonreactive water. Mass densities and probability distributions for the values of the energy gaps computed in the NPT ensemble reveal a negligible role of the reactive potential in the limit of low concentrated solutions, thus corroborating for the first time the validity of approximations based on the canonical NVT ensemble, customarily adopted for EVB simulations. The presented formalism also aims to contribute to future implementations and extensions of the EVB method to research the limit of highly concentrated solutions.

Vessel elements of two thelypteroid ferns-part I.

BACKGROUND: Hydraulically efficient xylem was evolved in the vascular plants as an apomorphy of the group. Main xylem components involved in water conduction are tracheid and vessel. Vessels, in which two ends are perforated, constituted major evolutionary innovation within vascular plants, presumably providing more efficient solute conduction. Not all vascular plants have vessels. In pteridophytes vessels are present only in seven genera. The contention lies regarding the presence and distribution of vessel in pteridophytes are the impulsive force of this investigation. METHODS: Tracheary elements are isolated following the standard maceration technique, then hand-razor cut longisections are passed through the aqueous alcohol grades and air-dried samples are placed on stub, sputter coated with gold and examined with SEM. RESULTS: Two thelypteroid ferns viz. Ampelopteris prolifera (Retz.) Copel. and Thelypteris interrupta (Willd.) K. Iwats. are having vessel elements in root, rhizome, stipe, rachis, primary vein/costa, root-rhizome and rhizome-petiole junction i.e. through entire vascular connection of the plant body though the vessel network is interrupted and joined with parenchyma at the end in some places. Presence of vessel elements in the costa of pteridophytic taxa is first time reported by this study. Vessel end-walls are obliquely placed (root, rhizome, and stipe) but oblique to horizontal orientation is noticed in the primary vein/costa. End-walls are with simple, intermediate and compound perforation plates observed through SEM imaging as well as with tissue specific stain. Studied taxa are grown either in terrestrial microclimate of two contrasting environments i.e. sun and shade (A. prolifera) or in open swampy land (T. interrupta) with moderate to highly disturbed places as rapid proliferating populations showing interpopulation variations of tracheary elements length-width(s) and vessel end-wall length-width(s). CONCLUSION: Vessel elements are present throughout the entire vascular connections of the plant body of A. prolifera (Retz.) Copel. and T. interrupta (Willd.) K. Iwats. Interpopulation variation of tracheary elements length-width(s) and vessel end-wall length width(s) are noticed. Till date only seven genera of pteridophytes are reported for the presence of vessel and these two genera are the new addition with the previous.

A QM/MM Study of Nitrite Binding Modes in a Three-Domain Heme-Cu Nitrite Reductase.

Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2-) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2- to the T2Cu centre, but very little is known about how NO2- binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2- may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2- with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Distinguishing Nitro vs Nitrito Coordination in Cytochrome c' Using Vibrational Spectroscopy and Density Functional Theory.

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c' (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (Kd ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe-NO2), δ(ONO), and νs(NO2) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO}6 and {FeNO}7 states, enabling FTIR measurements to distinguish νs(NO2) and νas(NO2) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H2O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of heme-nitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.

Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase.

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCAT and HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site 'capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.

Coupled Valence-Bond State Molecular Dynamics Description of an Enzyme-Catalyzed Reaction in a Non-Aqueous Organic Solvent.

Enzymes are widely used in nonaqueous solvents to catalyze non-natural reactions. While experimental measurements showed that the solvent nature has a strong effect on the reaction kinetics, the molecular details of the catalytic mechanism in nonaqueous solvents have remained largely elusive. Here we study the transesterification reaction catalyzed by the paradigm subtilisin Carlsberg serine protease in an organic apolar solvent. The rate-limiting acylation step involves a proton transfer between active-site residues and the nucleophilic attack of the substrate to form a tetrahedral intermediate. We design the first coupled valence-bond state model that simultaneously describes both reactions in the enzymatic active site. We develop a new systematic procedure to parametrize this model on high-level ab initio QM/MM free energy calculations that account for the molecular details of the active site and for both substrate and protein conformational fluctuations. Our calculations show that the reaction energy barrier changes dramatically with the solvent and protein conformational fluctuations. We find that the mechanism of the tetrahedral intermediate formation during the acylation step is similar to that determined under aqueous conditions, and that the proton transfer and nucleophilic attack reactions occur concertedly. We identify the reaction coordinate to be mostly due to the rearrangement of some residual water molecules close to the active site.

Excited-State Proton Transfer Can Tune the Color of Protein Fluorescent Markers.

We show by quantum mechanical/molecular mechanical (QM/MM) simulations that phenylbenzothiazoles undergoing an excited-state proton transfer (ESPT) can be used to probe protein binding sites. For 2-(2'-hydroxy-4'-aminophenyl)benzothiazole (HABT) bound to a tyrosine kinase, the absolute and relative intensities of the fluorescence bands arising from the enol and keto forms are found to be strongly dependent on the active-site conformation. The emission properties are tuned by hydrogen-bonding interactions of HABT with the neighboring amino acid T766 and with active-site water. The use of ESPT tuners opens the possibility of creating two-color fluorescent markers for protein binding sites, with potential applications in the detection of mutations in cancer cell lines.

Role of two alternate water networks in Compound I formation in P450eryF.

The P450eryF enzyme (CYP107A1) hydroxylates 6-deoxyerythronolide B to erythronolide B during erythromycin synthesis by Saccharopolyspora erythraea. In many P450 enzymes, a conserved "acid-alcohol pair" is believed to participate in the proton shuttling pathway for O2 activation that generates the reactive oxidant (Compound I, Cpd I). In CYP107A1, the alcohol-containing amino acid is replaced with alanine. The crystal structure of DEB bound to CYP107A1 indicates that one of the substrate hydroxyl groups (5-OH) may facilitate proton transfer during O2 activation. We applied molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) techniques to investigate substrate-mediated O2 activation in CYP107A1. In the QM/MM calculations, the QM region was treated by density functional theory, and the MM region was represented by the CHARMM force field. The MD simulations suggest the existence of two water networks around the active site, the one found in the crystal structure involving E360 and an alternative one involving E244. According to the QM/MM calculations, the first proton transfer that converts the peroxo to the hydroperoxo intermediate (Compound 0, Cpd 0) proceeds via the E244 water network with direct involvement of the 5-OH group of the substrate. For the second proton transfer from Cpd 0 to Cpd I, the computed barriers for the rate-limiting homolytic O-O cleavage are similar for the E360 and E244 pathways, and hence both glutamate residues may serve as proton source in this step.

A microiterative intrinsic reaction coordinate method for large QM/MM systems.

Intrinsic reaction coordinate (IRC) computations are a valuable tool in theoretical studies of chemical reactions, but they can usually not be applied in their current form to handle large systems commonly described by quantum mechanics/molecular mechanics (QM/MM) methods. We report on a development that tackles this problem by using a strategy analogous to microiterative transition state optimization. In this approach, the IRC equations only govern the motion of a core region that contains at least the atoms directly involved in the reaction, while the remaining degrees of freedom are relaxed after each IRC step. This strategy can be used together with any existing IRC procedure. The present implementation covers the stabilized Euler, local quadratic approximation, and Hessian predictor-corrector algorithms for IRC calculations. As proof of principle, we perform tests at the QM level on small gas-phase systems and validate the results by comparisons with standard IRC procedures. The broad applicability of the method is demonstrated by IRC computations for two enzymatic reactions using standard QM/MM setups.

Interfacial States in Donor-Acceptor Organic Heterojunctions: Computational Insights into Thiophene-Oligomer/Fullerene Junctions.

Donor-acceptor heterojunctions composed of thiophene oligomers and C60 fullerene were investigated with computational methods. Benchmark calculations were performed with time-dependent density functional theory. The effects of varying the density functional, the number of oligomers, the intermolecular distance, the medium polarization, and the chemical functionalization of the monomers were analyzed. The results are presented in terms of diagrams where the electronic states are classified as locally excited states, charge-transfer states, and delocalized states. The effects of each option for computational simulations of realistic heterojunctions employed in photovoltaic devices are evaluated and discussed.

Coupled electron transfer and proton hopping in the final step of CYP19-catalyzed androgen aromatization.

Aromatase (CYP19) catalyzes the terminal step in estrogen biosynthesis, which requires three separate oxidation reactions, culminating in an enigmatic aromatization that converts an androgen to an estrogen. A stable ferric peroxo (Fe(3+)O(2)(2-)) intermediate is seen by electron paramagnetic resonance, but its role in this complex reaction remains controversial. Combining molecular dynamics simulation and hybrid quantum mechanics/molecular mechanics, we show that ferric peroxo addition to the 19-aldehyde initiates the reaction. Stepwise cleavage of the C10-C19 and O-O bonds of the peroxohemiacetal extrudes formate and yields Compound II, which in turn desaturates the steroid through successive abstraction of the 1ß-hydrogen atom and deprotonation of the 2ß-position. Throughout the transformation, a proton is cyclically relayed between D309 and the substrate to stabilize reaction intermediates. This mechanism invokes novel oxygen intermediates and provides a unifying interpretation of past experimental mechanistic studies.

Peroxo-iron mediated deformylation in sterol 14alpha-demethylase catalysis.

The mechanisms of cytochrome P450 (CYP) catalyzed C-C bond cleavage have been strongly debated and difficult to unravel. Herein, deformylation mechanisms of the sterol 14alpha-demethylase (CYP51) from Mycobacterium tuberculosis are elucidated using molecular dynamics simulation, density functional theory, and hybrid quantum mechanics/molecular mechanics methods. These results provide strong theoretical support for the operation of the peroxo intermediate in CYP-catalyzed deformylation. Molecular dynamics simulations support the lanosterol carboxaldehyde intermediate diverts the hydrogen-bonded network of water putatively involved in proton delivery to peroxo and compound 0 (Cmpd 0) away from the O(2) ligand. In the presence of the aldehyde substrate, the peroxo intermediate is trapped as the peroxohemiacetal without an apparent barrier, which may then be protonated in the active site. The unprotonated peroxohemiacetal provides a branch point for a concerted deformylation mechanism; however, a stepwise mechanism initiated by cleavage of the C-C bond was found to be more energetically feasible. Population analyses of the peroxoformate/deformylated substrate complex indicate that heterolytic cleavage of the C-C bond in the enzyme environment generates a carbanion at C14. Conversely, in the absence of the protein electrostatic background, the C-C cleavage reaction proceeds homolytically, indicating that the active site environment exerts a strong modulatory effect on the electronic structure of this intermediate. If the peroxohemiacetal is protonated, this species preferentially expels formic acid through an O-O cleavage transition state. After expulsion of the formyl unit, both proton-independent and -dependent pathways converge to a complex containing compound II, which readily abstracts the 15alpha-hydrogen, thereby inserting the 14,15 double bond into the steroid skeleton. Parallel studies considering nucleophilic addition of Cmpd 0 to the aldehyde intermediate indicated that this reaction proceeds with high energetic barriers. Finally, the hydrogen atom abstraction and proton coupled electron transfer mechanism (J. Am. Chem. Soc. 2005, 127, 5224-5237) for compound I (Cmpd I) mediated deformylation of the geminal diol was considered in the context of the protein environment. In contrast to gas phase calculations, triradicaloid and pentaradicaloid Cmpd I states failed to initiate a concerted deformylation of the geminal diol. This study provides a unified mechanistic view consistent with decades of experiments aimed at understanding the deformylation reaction. Additionally, these results provide general mechanistic insight into the catalytic mechanisms of several biosynthetic and xenobiotic-oxidizing CYP enzymes of biomedical importance.

Molecular oxygen activation and proton transfer mechanisms in lanosterol 14alpha-demethylase catalysis.

The CYP51 lanosterol 14alpha-demethylases are evolutionarily ancient enzymes ubiquitously distributed throughout the biological domains. The experimental X-ray crystal structure of Mycobacterium tuberculosis (Mtb) CYP51 is the first of an enzyme capable of catalyzing inert C-C bond cleavage. Amino acid sequence comparisons of CYP51 family members with other members of the CYP superfamily reveal the almost universally conserved "acid-alcohol" pair, putatively involved in proton transport and O(2) activation, is replaced with a His-Thr dyad. In this study, extended molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics calculations (QM/MM) are applied to characterize reactive oxygen intermediates and to unravel mechanisms of O(2) activation vis-a-vis proton transport for this important enzyme. MD confirms stable His259deltaH(+)-Thr260OH-O(2) (Mtb numbering) hydrogen bonding early in the simulations, suggesting these amino acids could function similarly to the Asp251-Thr252 pair in CYP101. QM/MM calculations support this dyad competently catalyzes the peroxo to Compound 0 (Cmpd 0) reaction, albeit an endothermic homolytic O-O scission mechanism affording Compound I (Cmpd I) was identified. Disruption of the His259H(+)-Thr260OH hydrogen bond in MD simulation divulges a second previously unidentified hydrogen-bond network, including three water molecules linking Glu173 in the CYP51 F-helix to the distal O(2) atom. Expansion of the QM region to contain these atoms unveils an unprecedented triradicaloid electronic structure of the peroxo intermediate characterized by spin polarization to the Glu173 side chain, attributable to the protein electrostatic environment. This amino acid, in concert with an active-site water network, catalyzes a facile protonation of the peroxo intermediate and offers a series of redundant heterolytic and homolytic mechanisms, affording exothermic formation of the ultimate oxidant Cmpd I. In summary, this study highlights the importance of the protein electrostatic environment to tune the electronic structure of CYP catalytic intermediates in addition to Cmpd I and illustrates the diversity of proton transport pathways available to these enzymes to drive catalysis.