Hydrogenases are metalloenzymes that catalyze the cleavage and evolution of dihydrogen (H2), a perfectly clean fuel. Thus, they represent ideal model catalysts for sustainable energy conversion approaches utilizing H2. Due to the presence of biologically uncommon CO and CN- ligands at their catalytic metal sites, infrared (IR) spectroscopy is a key technique in hydrogenase research that can even be used to study these enzymes within living cells. Here, we introduce two-dimensional (2D) IR spectroscopy as a new in vivo technique for exploring the impact of the conditions in the cytoplasm on the properties of hydrogenases. Utilizing the soluble NAD+-reducing [NiFe] hydrogenase from the H2-oxidizing model bacterium Cupriavidus necator H16 as a suitable and biotechnologically relevant model enzyme, we demonstrate the feasibility of this approach. Our data indicates that even subtle structural details of the [NiFe] active site are unaffected by the unique gel-like properties of the highly dense cytoplasm, pointing towards a shielding role of the protein matrix that isolates this deeply buried metal center from environmental influences. In a more general sense, this study demonstrates that adequate strategies for scatter suppression can turn 2D-IR spectroscopy into a suitable technique for probing enzymes and other molecular targets in living cells and other complex biological environments.

The dynamic three-dimensional structures of proteins dictate their function, but accessing structures in solution at physiological temperatures is challenging. Ultrafast 2D-IR spectroscopy of the protein amide I band produces a spectral fingerprint that derives directly from the 3D backbone structure within minutes, using microlitres of label-free samples, in aqueous (H2O) solution and with picosecond time resolution. However, transforming 2D-IR fingerprints into quantitative, solution-phase protein structures relies on decoding the fundamental link between the atomistic structure and the 2D spectrum. We demonstrate a top-down approach to solution-phase protein structure determination that combines 2D-IR spectral libraries with machine learning (ML). Using a dataset consisting of 6732 spectra of 35 proteins in H2O that span a range of structures, Support-Vector Machine (SVM) models classified unknown protein samples according to structural content and measured quantities of α-helix and β-sheet with an RMS error of ≤7%. The potential for hybrid 2D-IR-ML tools to predict the number and length of helices in a protein, and identify the presence of parallel and antiparallel β-sheets from the 2D-IR fingerprint is also demonstrated. These results lay the groundwork for rapid, quantitative analysis of dynamic protein structures under physiologically relevant conditions.

Using ultrafast time-resolved infrared (TRIR) spectroscopy, we studied the solution-phase excited-state structural evolution of an indacenodithiophene-co-benzothiadiazole polymer (C8-IDTBT). Following band gap excitation, the TRIR spectra reveal vibrational features that develop within 10 ps and decay over 4 ns. Using pulse radiolysis measurements, charge-modulation spectroscopy, and quantum-chemical calculations, the IR features are assigned to polaron pairs. Interestingly, these features appear on an evolving broad mid-IR electronic absorption background, with kinetics correlating with the formation and decay of the cation-radical vibrational bands. A three-state kinetic model successfully reproduces the spectral evolution, revealing that the polaron and exciton populations exist in dynamic equilibrium on picosecond time scales, with time constants for exciton dissociation in the range of 3-5 ps and polaron-to-exciton reformation between 20 and 100 ps, while both species decay to the ground state on much slower nanosecond time scales (∼1 ns), yielding a remarkably high polaron-generation efficiency, higher than 50%. These findings provide fundamental insights into intramolecular charge photogeneration mechanisms in conjugated polymers, demonstrating efficient bound-polaron formation in isolated polymer chains.

[FeFe] hydrogenases catalyze the reversible cleavage of H2, a clean fuel, at exceptional rates. Therefore, understanding their catalytic mechanism is of high importance. Here, we employ multiscale UVpump-IRprobe spectroscopy to study the reversible photochemical activation of the CO-inhibited Hox-CO state over picosecond to millisecond time scales. Since this process transforms the catalytic site into the active, H2-binding Hox state, photolysis of Hox-CO represents a unique strategy for studying light-triggered activation and the catalytic cycle of the enzyme with high time resolution. We show that the extrinsic CO of Hox-CO dissociates in picoseconds and remains unbound for up to milliseconds. During this time, the enzyme is available for H2 binding and further catalytic transformations. This time window is sufficiently large to study catalytic processes from the earliest steps to completion of the catalytic cycle. Our approach provides a basis for investigating the catalytic cycle of [FeFe] hydrogenases in real time and without diffusion limitation.

Visible-light-activated radical photoinitiators are pivotal in the efficient construction of complex molecular architectures and the precision synthesis of advanced polymeric materials. At the heart of their function lies the formation of reactive radical species that drive selective homolytic bond cleavage, but elucidating the fundamental mechanisms of these processes is notoriously difficult due to the fleeting nature of key intermediates. In this study, time-resolved infrared (TRIR) spectroscopy provides a powerful window into the complete reaction profile of the versatile photocatalyst [Mn2(CO)10], including the observation of [Mn(O2)(CO)5], which is a long-lived (ms) reservoir of the reactive 17-electron complex [Mn(CO)5]. We give unprecedented structural and mechanistic insights concerning the formation of [Mn(CO)5] in electronically and vibrationally excited states (fs-ps), its quenching by O2 (ns), regeneration of the ground-state catalyst, and C-I bond activation (ms). New avenues for the rational design of next-generation metal-metal photocatalysts are provided, as [Mn(O2)(CO)5] significantly extends the catalyst longevity.

Acetyl-CoA synthase (ACS) catalyzes the condensation of acetyl-CoA from carbon monoxide (CO), a methyl group, and coenzyme A, enabling the fixation of CO into biomolecules. Recent low-temperature ENDOR studies proposed that the enzyme can bind two CO ligands in its reduced Ared-CO state, reshaping the view of CO coordination and inhibition of ACS. However, whether this two-CO model reflects a physiologically relevant state has remained an open question. To address this issue, we examined ACS under near-native, ambient conditions using ultrafast and two-dimensional infrared spectroscopy, complemented by anharmonic frequency calculations. These methods provide a wealth of structural and dynamical information beyond insights from conventional IR absorption spectroscopy, allowing a direct view of CO coordination in the Ared-CO state. Our results demonstrate that ACS binds a single CO ligand under ambient conditions. This finding clarifies the stoichiometry of CO coordination in ACS and underscores the broader potential of advanced IR spectroscopy, combined with computation, to unravel ligand binding in complex bioorganometallic systems.

ABSTRACT [FeFe] hydrogenases are highly active, reversible enzymes for the interconversion of hydrogen with protons and electrons. Their active site H‐cluster consists of a canonical [4Fe‐4S] cluster covalently linked to a unique [2Fe] H centre. Their catalytic mechanism has been studied extensively, but several details remain disputed, and two rival models exist in the literature. One crucial difference between these models is the structure and catalytic relevance of two states named H red H + and H sred H + . In the first model, these states are catalytic intermediates containing a reduced [Fe(I)Fe(I)] H centre and a bridging CO ligand (µCO), while in the second model they are inactive states containing an oxidised [Fe(II)Fe(II)] H site and a bridging hydride ligand (µH − ). The second proposal was initially based on the lack of a prominent absorption peak attributed to a µCO ligand in the infrared (IR) spectra of both states. Here, we provide evidence for the presence of a µCO ligand in the H red H + and H sred H + states using two‐dimensional (2D) IR spectroscopy, firmly establishing the structure of these states as [Fe(I)Fe(I)] H with a µCO ligand. The results suggest that these states are catalytically relevant intermediates with crucial implications for understanding hydrogen conversion in nature and designing new synthetic catalysts.

The synthesis and photophysical properties of four [Re(R-NI-phen)(CO)3Cl] complexes (Re-R), where NI = 1,8-naphthalimide and phen = 1,10-phenanthroline, are reported. The R-NI chromophores were substituted at their 4-positions with R = H (Re-H), ethynyl benzene (Re-EB), ethynyl thiophene (Re-ES), ethynyl trimethylsilane (Re-ET), yielding a series of metal-organic bichromophores. The R-NI-phen organic chromophores were investigated in parallel to better understand the photophysical properties exhibited in the Re-R complexes. The excited-state processes of these molecules were studied by transient absorption and time-resolved infrared spectroscopies, to unravel the kinetics of the energy transfer processes occurring in the Re-R series. Namely, the interplay between the singlet ligand-centered (1LC), the triplet metal-to-ligand charge-transfer (3MLCT), and the triplet ligand-centered (3LC) excited states were studied using these time-resolved spectroscopic techniques, supported by quantum chemical calculations, to monitor the changing excited-state dynamics in the Re-R series. These studies reveal that the thermal equilibrium between the 3MLCT and 3LC excited states present in Re-H is modulated in favor of the 3LC excited state in the remaining Re-R series, giving rise to increasingly ligand-like photophysics as the electron-donating strength of the R-group increases.

Hydrogenases are metalloenzymes that catalyze the reversible splitting of dihydrogen (H2), a clean and sustainable fuel. In this study, we investigate the reversible photodissociation and rebinding of an extrinsic carbon monoxide (CO) ligand at the active site of a [NiFe] model hydrogenase. CO acts as a catalytic inhibitor of the enzyme, whereas its photolysis restores an active state capable of H2 binding. Using UVpump-IRprobe spectroscopy in a multiple-probe configuration that allows covering picosecond to millisecond time scales, we characterize the reaction dynamics following CO photolysis. The results reveal a large temporal window between rapid CO dissociation and slow rebinding, enabling the detailed investigation of H2 binding and activation at the active site, unaffected by H2 mass transport limitation.

The dynamics of double-stranded DNA (dsDNA) are central to its biological role as a repository of genetic information. However, under physiological conditions DNA is subject to base mispairing and the formation of abasic sites through processes such as depurination. Such site-specific changes to the established Watson-Crick (WC) architecture would be expected to influence duplex dynamics and so affect key processes including protein binding. Here, we apply temperature-jump infrared spectroscopy to interrogate the relative impact of base pair mismatches and abasic sites on the structural dynamics of a 21-base pair (bp) dsDNA oligomer. The inclusion of an abasic site in the center of the strand leads to destabilization that is manifest as <1 μs time scale disruption of the nearby bases that is not present in fully WC-base paired sequences. Comparing this behavior with sequences featuring mismatches of different sizes shows that a single-bp mismatch causes minimal destabilization, whereas a triple base mismatch results in dynamics that closely mimic those resulting from the presence of an abasic site.

Photosensitized damage by the mechanism of direct 1e- transfer from a nucleobase to the metal complex is a complementary approach to type I and type II methods of photodynamic therapy. In this ultrafast spectroscopic study we report the ability of a nitrile infrared redox probe to report on the photo-oxidation of guanine-rich DNA, comprising persistent runs of guanine, by the dppz-10-CN containing complex [Ru(TAP)2(dppz-10-CN)]2+ (12+), dppz-10-CN = 10-cyano-dipyrido[3,2-a:2',3'-c]phenazine and TAP = 1,4,5,8-tetraazaphenanthrene. Our study reveals the ability of the enantiomers of 12+ to photo-oxidize guanine in double-stranded and quadruplex DNA. Transient visible absorption reveals a high yield of the formation of the photoreduced metal complex due to photo-oxidation of guanine in the quadruplex-bound 12+ systems, and that this is greater for the Λ enantiomer. Spectro-electrochemical and computational studies indicate the role of the dppz-10-CN as the preferred site of reduction, while time-resolved electronic absorption (TrA) spectroscopy highlights the impact of the enantiomers on the yield of photo-oxidation in the DNA systems. Notably, time-resolved infrared (TRIR) spectroscopy allows comprehensive tracking of the photo-oxidation dynamics by monitoring four key components, namely: (1) the transient band of the Ru/TAP-based lowest 3MLCT excited state, (2) bleach bands associated with DNA bases in close proximity to the excited state "site effect", (3) the guanine radical cation band at ca. 1700 cm-1 and (4) the amplification of the red-shifted nitrile stretching vibration of the transient dppz-reduced complex. Together, these results allow detailed profiling of photoinduced electron transfer in DNA-bound ruthenium(II) polypyridyl complex systems and highlight the potential of such redox probes. Overall, this study presents an important insight regarding the nature of charge transfer in a Hoogsteen-bound guanine quadruplex compared to Watson-Crick GC base pairings.

Tryptophan is the most efficient fluorophore of the naturally occurring amino acids and is widely used as a fluorescence probe of protein structure and function. As a result of its importance, there have been numerous studies of the ultrafast photochemical dynamics of tryptophan. Nonetheless, these studies have not identified the pathway to the triplet state, which competes with fluorescence emission. Here, we combine femtosecond-to-microsecond time-resolved transient absorption spectroscopy and time-resolved infrared spectroscopy to explore the photochemical pathway from the UV excitation of tryptophan in aqueous solution to the population of the triplet state and its subsequent relaxation. We observe prompt formation of cations and solvated electrons consistent with autoionization to form a cation-electron ion pair. We find that the cation-electron ion pair subsequently decays with time scales that match the fluorescence lifetime of tryptophan in aqueous solution, indicative of a dynamic equilibrium between the fluorescent state and the cation-electron ion pair. We also find that population of the triplet state occurs on the same time scale as the decay of the cation-electron ion pair and fluorescence, indicating that the triplet state is populated either by recombination of a separated cation and electron after a spin flip or by intersystem crossing from the fluorescent state. Regardless of which mechanism dominates, population of the triplet state of tryptophan is governed by the dynamic equilibrium between the fluorescent state and the cation-electron ion pair.

The design of efficient molecular devices that convert heat into electricity requires an understanding of how to control electrical and thermal conductance. While the electrical conductance of π-conjugated systems has been widely studied, its thermal counterpart is much less explored. In this work, we use pump-probe infrared spectroscopy and theoretical modelling to study heat transport in unsubstituted and alkyl chain-substituted π-conjugated fluorenyl derivatives. Our findings indicate that decoration with alkyl chains increases both the rate of intramolecular and intermolecular heat transport by increasing the number of pathways for heat propagation and dissipation, respectively. These results provide design rules for controlling thermal conductance in conjugated molecular systems.

Reversibly switchable fluorescent proteins (rsFPs) are essential tools in super-resolution imaging. The mechanism operating in the widely applied negative switching rsFPs has been studied in detail. Much less attention has been paid to the positive switching rsFP variants, which offer the potential benefit of emissive states that do not photoswitch during measurement. Here we probe photochemical mechanism in all three photoactive states of the positive switching rsFP Kohinoor using a combination of ultrafast transient absorption, to probe chromophore population dynamics, and time resolved infrared, to access both chromophore populations and their effect on the surrounding protein matrix. We establish that none of the photochemical reactions are simple rate processes with transient absorption and transient IR data characterised by a common two component relaxation mechanism. Transient IR measurements reveal instantaneous coupling between the electronically excited chromophore and its protein environment, indicating that coupling arises from electrostatic or H-bonded interactions. In both on- and off-switching states the early phase of the excited state dynamics involve an initial relaxation in the perturbed protein environment, which leads to an intermediate state from which chromophore isomerization occurs. This result suggests that the protein dynamics play an active role in steering the excited state reaction, which is in-turn consistent with the known key role of the protein environment in tuning FP photophysics. Identifying and modifying the interactions between chromophore and protein will provide a means to optimise rsFP performance and thus a basis for development of improved labels for super-resolution bioimaging.

Dreiklang is a reversibly switchable (rs) fluorescent protein (FP) with a unique off-state, a UV absorbing hydrated form of the typical FP chromophore. Here we report ultrafast dynamics of the off- to on-state transition in Dreiklang using complementary ultrafast optical and vibrational transient absorption to resolve chromophore driven protein structural dynamics. This approach allows observation of the real-time response in a protein to bond breaking and forming events. The excited electronic state decays in a nonsingle exponential fashion in tens to hundreds of picoseconds, undergoing photodehydration with a yield of several per-cent. The primary photoproduct formed is identified as the cis protonated form of the FP chromophore, initially in a perturbed H-bonded environment. This primary product relaxes on a few microseconds timescale by a mechanism involving changes to a glutamic acid residue and modifications of the amide backbone, possibly involving a carbonyl to imine tautomerization. The temporal and spectral resolution of Dreiklang's photodehydration provides data against which to test quantum chemical calculations of reaction dynamics in proteins and suggests a route to modifying and potentially enhancing its photoswitching properties.

We investigated ultrafast defect-lattice dynamics in diamond using the N s : H − C 0 defect, an analog of bond-centered hydrogen in semiconductors. Combining synthesis, ultrafast vibrational spectroscopy, and calculations, we show that excitation of the defect’s stretch mode leads to the generation of localized phonons and the formation of a hot ground state, where the interatomic potential is transiently modified. Our results reveal unexpected nonequilibrium phonon effects despite diamond’s exceptionally high thermal conductivity, with implications for quantum defect engineering.

The ultrafast photophysics of many isomerizing molecules involves subpicosecond formation of a twisted hot ground state, which transfers energy to the environment through vibrational relaxation (cooling) over several picoseconds. In time-resolved infrared (TR-IR) spectroscopy, hot ground state transients show frequency shifts and band reshapings, which cannot be described through kinetic models that assume static spectral functions. We report a simple anharmonic cascade framework, which uses a single adjustable parameter associated with scaling the probability of vibrational energy transfer to the environment, for describing hot ground state cooling (HGSC) in TR-IR spectroscopy. The model is demonstrated against measurements on the cyan fluorescent protein chromophore. To best describe HGSC band shape evolution, the model utilizes ab initio data on anharmonic vibrational structure and nonadiabatic molecular dynamics trajectories of S1→ S0 internal conversion for realistic vibration occupation numbers of the nascent hot ground state. The modeling framework is readily extended to include mode-specific rates for intermolecular energy transfer and can be applied to any ultrafast isomerizing molecule for which anharmonic vibrational properties can be computed.

An investigation into the effect of a phosphine coligand on the activation of precatalysts for manganese-catalyzed C-H bond functionalization is reported. Although simple precatalysts [MnBr(CO)5] and [Mn2(CO)10] are used extensively in these reactions, there is a dearth of alternate precatalyst structures, which has hindered the development of structure-activity relationships. In this work, the effect of substituting a carbonyl ligand for a phosphine ligand is reported. Investigation of the photochemical activation of the precatalyst fac-[Mn(inpy)(CO)3(PPh3)] (inpy = cyclometalated 1-(pyridin-2-yl)-1H-indole) 3 by time-resolved infrared spectroscopy (TRIR) reveals that light-induced dissociation of a CO ligand occurs preferentially over loss of the phosphine. The ultrafast dynamics of the initially formed solvent complex [Mn(inpy)(CO)2(toluene)(PPh3)] 9 are described, as is the slower substitution of the coordinated solvent by added pyridine to give [Mn(inpy)(CO)2(NC5H5)(PPh3)] 10. Replacing the pyridine with phenylacetylene again results in the substitution of the metal-bound toluene to give the alkyne complex [Mn(inpy)(η2-HC2Ph)(CO)2(PPh3)] 12. The alkyne undergoes a migratory insertion reaction into the Mn-C bond on a microsecond time scale with a very similar first-order rate constant to [Mn(inpy)(CO)4], 2, demonstrating that this key step in Mn-catalyzed reactions is not affected by the presence of the phosphine ligand.

[FeFe] hydrogenases are Nature's most efficient catalysts for the cleavage and evolution of molecular hydrogen. Despite decades of research, key aspects of the catalytic cycle and the underlying geometrical and electronic properties of the active-site cofactor, called the H-cluster, are not fully understood. Spectroscopic techniques have played a central role in establishing the current state of knowledge on [FeFe] hydrogenases, and further advances in the field depend critically on novel techniques that yield so-far inaccessible insights into structural and mechanistic aspects. Infrared (IR) absorption spectroscopy represents a well-established and versatile technique that can identify and characterize all active and inactive states of the H-cluster by means of structurally sensitive and spectrally isolated CO and CN stretching vibrations. However, the amount of information that can be extracted from these linear experiments is inherently limited. Here we introduce experimental and computational two-dimensional (2D-)IR spectroscopy for the characterization of [FeFe] hydrogenases. Utilizing the Hinact state of the H-cluster as a model system, we demonstrate that this nonlinear technique yields direct information about the nature and interactions of the CO and CN stretching vibrations. These insights allow, for the first time, to quantitatively describe the character of these widely used reporter vibrations, their spatial localization, and the way they change upon structural variation of the H-cluster. The strength of this approach is demonstrated by correctly identifying the proposed structure of the Hinact state, in solution and at ambient temperature. In conclusion, the introduced combination of experimental and computational 2D-IR spectroscopy represents a powerful approach for studying [FeFe] hydrogenases and other complex organometallic targets.

OaPAC, the photoactivated adenylyl cyclase from Oscillatoria acuminata, is composed of a blue light using FAD (BLUF) domain fused to an adenylate cyclase (AC) domain. Since both the BLUF and AC domains are part of the same protein, OaPAC is a model for understanding how the ultrafast modulation of the chromophore binding pocket caused by photoexcitation results in the activation of the output domain on the μs-s time scale. In the present work, we use unnatural amino acid mutagenesis to identify specific sites in the protein that are involved in transducing the signal from the FAD binding site to the ATP binding site. To provide insight into site-specific structural dynamics, we replaced W90 which is close to the chromophore pocket, F103 which interacts with W90 across the dimer interface, and F180 in the central core of the AC domain, with the infrared probe azido-Phe (AzPhe). Using ultrafast IR, we show that AzPhe at position 90 responds on multiple time scales following photoexcitation. In contrast, the light minus dark IR spectrum of AzPhe103 shows only a minor perturbation in environment between the dark and light states, while replacement of F180 with AzPhe resulted in a protein with no catalytic activity. We also replaced Y125, which hydrogen bonds with N256 across the dimer interface, with fluoro-Tyr residues. All the fluoro-Tyr substituted proteins retained the light-induced red shift in the flavin absorption spectrum; however, only the 3-FY125 OaPAC retained photoinduced catalytic activity. The loss of activity in 3,5-F2Y125 and 2,3,5-F3Y125 OaPAC, which potentially increase the acidity of the Y125 phenol by more than 1000-fold, suggests that deprotonation of Y125 disrupts the signal transduction pathway from the BLUF to the AC domain.