The induced fit binding model describes a conformational change occurring when a small molecule binds to its biomacromolecular target. The result is enhanced noncovalent interactions between the ligand and biomolecule. Induced fit is well-established for small molecule–protein interactions, but its relevance to small molecule–DNA binding is less clear. We investigate the molecular determinants of Hoechst33258 binding to its preferred A-tract sequence relative to a suboptimal alternating A-T sequence. Results from two-dimensional infrared spectroscopy, which is sensitive to H-bonding and molecular structure changes, show that Hoechst33258 binding results in loss of the minor groove spine of hydration in both sequences, but an additional perturbation of the base propeller twists occurs in the A-tract binding region. This induced fit maximizes favorable ligand–DNA enthalpic contributions in the optimal binding case and demonstrates that controlling the molecular details that induce subtle changes in DNA structure may hold the key to designing next-generation DNA-binding molecules.

Changes in the structural and solvation dynamics of a 15mer AT DNA duplex upon melting of the double-helix are observed by a combination of ultrafast two-dimensional infrared (2D-IR) and optical Kerr-effect (OKE) spectroscopies. 2D-IR spectroscopy of the vibrational modes of the DNA bases reveal signature off-diagonal peaks arising from coupling and energy transfer across Watson–Crick paired bases that are unique to double-stranded DNA (ds-DNA). Spectral diffusion of specific base vibrational modes report on the structural dynamics of the duplex and the minor groove, which is predicted to contain a spine of hydration. Changes in these dynamics upon melting are assigned to increases in the degree of mobile solvent access to the bases in single-stranded DNA (ss-DNA) relative to the duplex. OKE spectra exhibit peaks that are assigned to specific long-range phonon modes of ds- and ss-DNA. Temperature-related changes in these features correlate well with those obtained from the 2D-IR spectra although the melting temperature of the ds-DNA phonon band is slightly higher than that for the Watson–Crick modes, suggesting that a degree of long-range duplex structure survives the loss of Watson–Crick hydrogen bonding. These results demonstrate that the melting of ds-DNA disrupts helix-specific structural dynamics encompassing length scales ranging from mode delocalisation in the Watson–Crick base pairs to long-range phonon modes that extend over multiple base pairs and which may play a role in molecular recognition of DNA.

Antimicrobial resistance represents a growing global health problem. The emergence of novel resistance mechanisms necessitates the development of alternative approaches to investigate the molecular fundamentals of resistance, leading ultimately to new strategies for counteracting them. To gain deeper insight into antibiotic–target interactions, the binding of the frontline anti-tuberculosis drug isoniazid (INH) to a target enzyme, InhA, from Mycobacterium tuberculosis was studied using ultrafast two-dimensional infrared (2D-IR) spectroscopy and molecular simulations. Comparing wild-type InhA with a series of single point mutations, it was found that binding of the INH–NAD inhibitor to susceptible forms of the enzyme increased the vibrational coupling between residues located in the Rossmann fold co-factor binding site of InhA and suppressed dynamic fluctuations of the enzyme structure. The effect correlated with biochemical assay data, being reduced in the INH-resistant S94A mutant and absent in the biochemically-inactive P193A control. Molecular dynamics simulations and calculations of inter–residue couplings indicate that the changes in coupling and dynamics are not localised to the co-factor binding site, but permeate much of the protein. We thus propose that the resistant S94A mutation circumvents subtle changes in global structural dynamics caused by INH upon binding to the wild-type enzyme that may impact upon the formation of important protein–protein complexes in the fatty acid synthase pathway of M. tuberculosis.

This article reviews the application of transient techniques in the elucidation of electron, proton, and photon chemistry related to the catalytic subsite of [FeFe] hydrogenase from the perspective of research in this area carried out at the UEA and Strathclyde laboratories. The detection of mixed-valence states, bridging CO intermediates, paramagnetic hydrides, and coordinatively unsaturated species has both informed understanding of biological catalysis and stimulated the search for stable analogues of key structural motifs likely involved in turnover states.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A–T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar–phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson–Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA.

Encapsulation of subsite analogues of the [FeFe]-hydrogenase enzymes in supramolecular structures has been shown to dramatically increase their catalytic ability, but the molecular basis for this enhancement remains unclear. We report the results of experiments employing infrared absorption, ultrafast infrared pump–probe, and 2D-IR spectroscopy to investigate the molecular environment of Fe2(pdt)(CO)6 (pdt: propanedithiolate) [1] encapsulated in the dispersed alkane phase of a heptane–dodecyltrimethylammonium bromide–water microemulsion. It is demonstrated that 1 is partitioned between two molecular environments, one that closely resembles bulk heptane solution and a second that features direct hydrogen-bonding interactions with water molecules that penetrate the surfactant shell. Our results demonstrate that the extent of water access to the normally water-insoluble subsite analogue 1 can be tuned with micelle size, while IR spectroscopy provides a straightforward tool that can be used to measure and fine-tune the chemical environment of catalyst species in self-assembled structures.

Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H2O and D2O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump–probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.

Biomimetic compounds based upon the active subsite of the [FeFe]-hydrogenase enzyme system have been the focus of much attention as catalysts for hydrogen production: a clean energy vector. Until recently, use of hydrogenase subsite systems for light-driven hydrogen production has typically required the involvement of a photosensitizer, but the molecule [(μ-pdt)(μ-H)Fe2(CO)4(dppv)]+, (1; dppv = cis-1,2-C2H2(PPh2)2; pdt = 1,3-propanedithiolate) has been reported to catalyze the evolution of hydrogen gas under sensitizer-free conditions. Establishing the molecular mechanism that leads to photohydrogen production by 1 is thus an important step that may enable further development of this family of molecules as solar fuel platforms. Here, we report ultrafast UVpump–IRprobe spectroscopy of 1 at three different excitation wavelengths and in a range of solvents, including under the conditions required for H2 production. Combining spectroscopic measurements of the photochemistry and vibrational relaxation dynamics of 1 with ground-state density functional theory (DFT) calculations shows that, irrespective of experimental conditions, near-instantaneous carbonyl ligand loss is the main photochemical channel. No evidence for a long-lived excited electronic state was found. These results provide the first time-resolved data for the photochemistry of 1 and offer an alternative interpretation of the underlying mechanism of light-driven hydrogen generation.

Determining the chemical and structural modifications occurring within a protein during fundamental processes such as ligand or substrate binding is essential to building up a complete picture of biological function. Currently, significant unanswered questions relate to the way in which protein structural dynamics fit within the structure–function relationship and to the functional role, if any, of bound water molecules in the active site. Addressing these questions requires a multidisciplinary approach and complementary experimental techniques that, in combination, enhance our understanding of the complexities of protein chemistry. We exemplify this philosophy by applying both physical and biological approaches to investigate the active site chemistry that contributes to the inhibition of the Corynebacterium glutamicum catalase enzyme by nitric oxide. Ultrafast two-dimensional infrared spectroscopy (2D-IR) experiments exploit the NO ligand as a local probe of the active site molecular environment and shows that catalase displays a dynamically-restricted, ‘tight,’ structure. X-ray crystallography studies of C. glutamicum catalase confirm the presence of a conserved chain of hydrogen-bonded bound water molecules that link the NO ligand and the protein scaffold. This combination of bound water and restricted dynamics stands in stark contrast to other haem proteins, such as myoglobin, that exhibit ligand transport functionality despite the presence of a similar distal architecture in close proximity to the ligand. We conclude not only that the bound water molecules in the catalase active site play an important role in molecular recognition of NO but also may be part of the mechanistic operation of this important enzyme.

The development of multidimensional spectroscopic tools capable of resolving site-specific information about proteins and enzymes in the solution phase is an important aid to our understanding of biomolecular mechanisms, structure, and dynamics. Nicotinamide adenine dinucleotide (NAD) is a common biological substrate and so offers significant potential as an intrinsic vibrational probe of protein–ligand interactions but its complex molecular structure and incompletely characterized infrared spectrum currently limit its usefulness. Here, we report the FTIR spectroscopy of the oxidized and reduced forms of NAD at a range of pD values that relate to the “folded” and “unfolded” forms of the molecules that exist in solution. Comparisons with structural analogs and the use of density functional theory simulations provide a full assignment of the observed modes and their complex pD dependencies. Finally, ultrafast two-dimensional infrared spectra of the oxidized and reduced forms of NAD are reported and their usefulness as biomolecular probes is discussed.

A [FeFe]-hydrogenase model compound (µ-S(CH2)3S)Fe2(CO)4(PMe3)2 [1] has been encapsulated in a low molecular weight (LMW) hydrogelator (Fmoc–Leu–Leu). Linear infrared absorption spectroscopy, gel melting and ultrafast time-resolved infrared spectroscopy experiments reveal significant contrasts in chemical environment and photochemistry between the encapsulated molecules and solution phase systems. Specifically, the gel provides a more rigid hydrogen bonding environment, which restricts isomerisation following photolysis while imparting significant increases in stability relative to a similarly aqueous solution. Since understanding and ultimately controlling the mechanistic role of ligands near Fe centres is likely to be crucial in exploiting artificial hydrogenases, these gels may offer a new option for future materials design involving catalysts.

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Changes in the structural and solvation dynamics of a 15mer AT DNA duplex upon melting of the double-helix are observed by a combination of ultrafast two-dimensional infrared (2D-IR) and optical Kerr-effect (OKE) spectroscopies. 2D-IR spectroscopy of the vibrational modes of the DNA bases reveal signature off-diagonal peaks arising from coupling and energy transfer across Watson–Crick paired bases that are unique to double-stranded DNA (ds-DNA). Spectral diffusion of specific base vibrational modes report on the structural dynamics of the duplex and the minor groove, which is predicted to contain a spine of hydration. Changes in these dynamics upon melting are assigned to increases in the degree of mobile solvent access to the bases in single-stranded DNA (ss-DNA) relative to the duplex. OKE spectra exhibit peaks that are assigned to specific long-range phonon modes of ds- and ss-DNA. Temperature-related changes in these features correlate well with those obtained from the 2D-IR spectra although the melting temperature of the ds-DNA phonon band is slightly higher than that for the Watson–Crick modes, suggesting that a degree of long-range duplex structure survives the loss of Watson–Crick hydrogen bonding. These results demonstrate that the melting of ds-DNA disrupts helix-specific structural dynamics encompassing length scales ranging from mode delocalisation in the Watson–Crick base pairs to long-range phonon modes that extend over multiple base pairs and which may play a role in molecular recognition of DNA.

Determining the chemical and structural modifications occurring within a protein during fundamental processes such as ligand or substrate binding is essential to building up a complete picture of biological function. Currently, significant unanswered questions relate to the way in which protein structural dynamics fit within the structure–function relationship and to the functional role, if any, of bound water molecules in the active site. Addressing these questions requires a multidisciplinary approach and complementary experimental techniques that, in combination, enhance our understanding of the complexities of protein chemistry. We exemplify this philosophy by applying both physical and biological approaches to investigate the active site chemistry that contributes to the inhibition of the Corynebacterium glutamicum catalase enzyme by nitric oxide. Ultrafast two-dimensional infrared spectroscopy (2D-IR) experiments exploit the NO ligand as a local probe of the active site molecular environment and shows that catalase displays a dynamically-restricted, ‘tight,’ structure. X-ray crystallography studies of C. glutamicum catalase confirm the presence of a conserved chain of hydrogen-bonded bound water molecules that link the NO ligand and the protein scaffold. This combination of bound water and restricted dynamics stands in stark contrast to other haem proteins, such as myoglobin, that exhibit ligand transport functionality despite the presence of a similar distal architecture in close proximity to the ligand. We conclude not only that the bound water molecules in the catalase active site play an important role in molecular recognition of NO but also may be part of the mechanistic operation of this important enzyme.

A [FeFe]-hydrogenase model compound (µ-S(CH2)3S)Fe2(CO)4(PMe3)2 [1] has been encapsulated in a low molecular weight (LMW) hydrogelator (Fmoc–Leu–Leu). Linear infrared absorption spectroscopy, gel melting and ultrafast time-resolved infrared spectroscopy experiments reveal significant contrasts in chemical environment and photochemistry between the encapsulated molecules and solution phase systems. Specifically, the gel provides a more rigid hydrogen bonding environment, which restricts isomerisation following photolysis while imparting significant increases in stability relative to a similarly aqueous solution. Since understanding and ultimately controlling the mechanistic role of ligands near Fe centres is likely to be crucial in exploiting artificial hydrogenases, these gels may offer a new option for future materials design involving catalysts.

Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H2O and D2O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump–probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.