Previous studies of a thermophilic alcohol dehydrogenase (ht-ADH) demonstrated a range of discontinuous transitions at 30 °C that include catalysis, kinetic isotope effects, protein hydrogen-deuterium exchange rates, and intrinsic fluorescence properties. Using the Förster resonance energy transfer response from a Trp-NADH donor-acceptor pair in T-jump studies of ht-ADH, we now report microsecond protein motions that can be directly related to active site chemistry. Two distinctive transients are observed: a slow, kinetic process lacking a temperature break, together with a faster transient that is only detectable above 30 °C. The latter establishes a link between enzyme activity and microsecond protein motions near the cofactor binding site, in a region distinct from a previously detected protein network that communicates with the substrate binding site. Though evidence of direct dynamical links between microsecond protein motions and active site bond cleavage events is extremely rare, these studies highlight the potential of T-jump measurements to uncover such properties.

For small molecule reaction kinetics, computed reaction coordinates often mimic experimentally measured observables quite accurately. Although nowadays simulated and measured biomolecule kinetics can be compared on the same time scale, a gap between computed and experimental observables remains. Here we directly compared temperature-jump experiments and molecular dynamics simulations of protein folding dynamics using the same observable: the time-dependent infrared spectrum. We first measured the stability and folding kinetics of the fastest-folding β-protein, the GTT35 WW domain, using its structurally specific infrared spectrum. The relaxation dynamics of the peptide backbone, β-sheets, turn, and random coil were measured independently by probing the amide I′ region at different frequencies. Next, the amide I′ spectra along folding/unfolding molecular dynamics trajectories were simulated by accurate mixed quantum/classical calculations. The simulated time dependence and spectral amplitudes at the exact experimental probe frequencies provided relaxation and folding rates in agreement with experimental observations. The calculations validated by experiment yield direct structural evidence for a rate-limiting reaction step where an intermediate state with either the first or second hairpin is formed. We show how folding switches from a more homogeneous (apparent two-state) process at high temperature to a more heterogeneous process at low temperature, where different parts of the WW domain fold at different rates.

Small fast folding subdomains with low contact order have been postulated to facilitate the folding of larger proteins. We have tested this idea by determining how the fastest folding linear β-hairpin, CLN025, which folds on the nanosecond time scale, folds within the context of a two-hairpin WW domain system, which folds on the microsecond time scale. The folding of the wild type FBP28 WW domain was compared to constructs in which each of the loops was replaced by CLN025. A combination of FTIR spectroscopy and laser-induced temperature-jump coupled with infrared spectroscopy was used to probe changes in the peptide backbone. The relaxation dynamics of the β-sheets and β-turn were measured independently by probing the corresponding bands assigned in the amide I region. The folding rate of the CLN025 β-hairpin is unchanged within the larger protein. Insertion of the β-hairpin into the second loop results in an overall stabilization of the WW domain and a relaxation lifetime five times faster than the parent WW domain. In both mutants, folding is initiated in the turns and the β-sheets form last. These results demonstrate that fast folding subdomains can be used to speed the folding of more complex proteins, and that the folding dynamics of the subdomain is unchanged within the context of the larger protein.

DNAzymes are catalytic oligonucleotides with important applications in gene regulation, DNA computing, responsive soft materials, and ultrasensitive metal-ion sensing. The most significant challenge for using DNAzymes in vivo pertains to non-toxic delivery and maintaining function inside cells. We synthesized multivalent deoxyribozyme ‘10–23’ gold nanoparticle (DzNP) conjugates, varying DNA density, linker length, enzyme orientation, and linker composition in order to study the role of the steric environment and gold surface chemistry on catalysis. DNAzyme catalytic efficiency was modulated by steric packing and proximity of the active loop to the gold surface. Importantly, the 10–23 DNAzyme was asymmetrically sensitive to the gold surface, and when anchored through the 5′ terminus was inhibited 32-fold. This property was used to generate DNAzymes whose catalytic activity is triggered by thiol displacement reactions or by photoexcitation at λ = 532 nm. Importantly, cell studies revealed that DzNPs are less susceptible to nuclease degradation, readily enter mammalian cells, and catalytically down-regulate GDF15 gene expression levels in breast cancer cells, thus addressing some of the key limitations in the adoption of DNAzymes for in vivo work.

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe-4S] electron relay covalently attached to the H2binding site and a diiron cluster ligated by CO, CN-, and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe-4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe-4S]Hbut prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

Time-resolved step-scan Fourier transform infrared (FT-IR) spectroscopy has been shown to be invaluable for studying excited-state structures and dynamics in both biological and inorganic systems. Despite the established utility of this method, technical challenges continue to limit the data quality and more wide ranging applications. A critical problem has been the low laser repetition rate and interferometer stepping rate (both are typically 10 Hz) used for data acquisition. Here we demonstrate significant improvement in the quality of time-resolved spectra through the use of a kHz repetition rate laser to achieve kHz excitation and data collection rates while stepping the spectrometer at 200 Hz. We have studied the metal-to-ligand charge transfer excited state of Ru(bipyridine)3Cl2 in deuterated acetonitrile to test and optimize high repetition rate data collection. Comparison of different interferometer stepping rates reveals an optimum rate of 200 Hz due to minimization of long-term baseline drift. With the improved collection efficiency and signal-to-noise ratio, better assignments of the MLCT excited-state bands can be made. Using optimized parameters, carbonmonoxy myoglobin in deuterated buffer is also studied by observing the infrared signatures of carbon monoxide photolysis upon excitation of the heme. We conclude from these studies that a substantial increase in performance of ss-FT-IR instrumentation is achieved by coupling commercial infrared benches with kHz repetition rate lasers.

Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

Metal-ligand cooperativity is an essential feature of bioinorganic catalysis. The design principles of such cooperativity in metalloenzymes are underexplored, but are critical to understand for developing efficient catalysts designed with earth abundant metals for small molecule activation. The simple substrate requirements of reversible proton reduction by the [NiFe]-hydrogenases make them a model bioinorganic system. A highly conserved arginine residue (R355) directly above the exogenous ligand binding position of the [NiFe]-catalytic core is known to be essential for optimal function because mutation to a lysine results in lower catalytic rates. To expand on our studies of soluble hydrogenase-1 from Pyrococcus furiosus (Pf SH1), we investigated the role of R355 by site-directed-mutagenesis to a lysine (R355K) using infrared and electron paramagnetic resonance spectroscopic probes sensitive to active site redox and protonation events. It was found the mutation resulted in an altered ligand binding environment at the [NiFe] centre. A key observation was destabilization of the Nia3+-C state, which contains a bridging hydride. Instead, the tautomeric Nia+-L states were observed. Overall, the results provided insight into complex metal-ligand cooperativity between the active site and protein scaffold that modulates the bridging hydride stability and the proton inventory, which should prove valuable to design principles for efficient bioinspired catalysts. This journal is

Electron bifurcation, or the coupling of exergonic and endergonic oxidation-reduction reactions, was discovered by Peter Mitchell and provides an elegant mechanism to rationalize and understand the logic that underpins the Q cycle of the respiratory chain. Thought to be a unique reaction of respiratory complex III for nearly 40 years, about a decade ago Wolfgang Buckel and Rudolf Thauer discovered that flavin-based electron bifurcation is also an important component of anaerobic microbial metabolism. Their discovery spawned a surge of research activity, providing a basis to understand flavin-based bifurcation, forging fundamental parallels with Mitchell's Q cycle and leading to the proposal of metal-based bifurcating enzymes. New insights into the mechanism of electron bifurcation provide a foundation to establish the unifying principles and essential elements of this fascinating biochemical phenomenon.

We report photo-catalytic H2 production by hydrogenase (H2ase)-quantum dot (QD) hybrid assemblies. Quenching of the CdTe exciton emission is observed, consistent with electron transfer from quantum dot to H2ase. GC analysis shows light driven H2 production in the presence of a sacrificial electron donor with an efficiency of 4%, which is likely a lower limit to these hybrid systems. FTIR was employed for direct observation of active site reduction in unprecedented detail for photo-driven H2ase catalysis with sensitivity towards both H2ase and sacrificial electron donor. Photosensitization with Ru(bpy)32+ shows distinct FTIR photo- reduction properties generating all states along the steady-state catalytic cycle with minimal H2 production indicating slow, sequential one electron reduction steps. Comparing H2ase activity and FTIR results of both systems shows that QDs bind more efficiently for electron transfer and the final enzyme state is different for the two sensitizers. The possible origins of these differences and their implications for the enzymatic mechanism are discussed.