One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Methanotrophic bacteria catalyze the aerobic oxidation of methane to methanol using Cu-containing enzymes, thereby exerting a modulating influence on the global methane cycle. To facilitate the acquisition of Cu ions, some methanotrophic bacteria secrete small modified peptides known as “methanobactins,” which strongly bind Cu and function as an extracellular Cu recruitment relay, analogous to siderophores and Fe. In addition to Cu, methanobactins form complexes with other late transition metals, including the Group 12 transition metals Zn, Cd, and Hg, although the interplay among solution-phase configurations, metal interactions, and the spectroscopic signatures of methanobactin-metal complexes remains ambiguous. In this study, the complexation of Zn, Cd, and Hg by methanobactin from Methylocystis sp. strain SB2 was studied using a combination of absorbance, fluorescence, extended x-ray absorption fine structure (EXAFS) spectroscopy, and time-dependent density functional theory (TD-DFT) calculations. We report changes in sample absorbance and fluorescence spectral dynamics, which occur on a wide range of experimental timescales and characterize a clear stoichiometric complexation dependence. Mercury L3-edge EXAFS and TD-DFT calculations suggest a linear model for HgS coordination, and TD-DFT suggests a tetrahedral model for Zn2+ and Cd2+. We observed an enhancement in the fluorescence of methanobactin upon interaction with transition metals and propose a mechanism of complexation-hindered isomerization drawing inspiration from the wild-type Green Fluorescent Protein active site. Collectively, our results represent the first combined computational and experimental spectroscopy study of methanobactins and shed new light on molecular interactions and dynamics that characterize complexes of methanobactins with Group 12 transition metals.

Hydrogenase enzymes are nature’s solution to using molecular hydrogen as an energy source. While the air-sensitivity of these biological catalysts make them impractical for industrial hydrogen generation, synthetic mimics show promise in acting as effective substitutes. Hydrogenase model compounds also enable detailed investigation of the ultrafast dynamics and chemical reaction mechanisms using ultrafast infrared spectroscopy. This chapter reviews the progress in applying ultrafast transient infrared absorption and multidimensional spectroscopy to a range of small molecule hydrogenase model compounds, as well as several macromolecular and larger constructs. The rich vibrational structure and straightforward chemical modularity of these diiron compounds represent an exciting class of molecules that are, at the same time, excellent model systems for fundamental chemical dynamics, and practical molecular catalytic components with direct application to next-generation hydrogen-based energy strategies.

Within the conceptual framework of Redfield theory, the optical response function arises from the dynamical evolution of the system's density operator, where nonunitary relaxation is encoded in the Redfield relaxation superoperator. In the conventional approach, the so-called secular approximation neglects terms that induce transitions between distinct coherences and among coherences and populations. The rationale is that these nonsecular terms are small in comparison to the far more dominant population relaxation and coherence dephasing contributions. Since two-dimensional infrared (2D-IR) spectroscopy has significant contributions arising from population relaxation and transfer pathways, it can be challenging to isolate signatures of the nonsecular relaxation. We report here that in three diiron dithiolate hexacarbonyl complexes that serve as small-molecule models of the [FeFe] hydrogenase H-cluster subsite, a fortuitous vibrational energy structure enables direct and clear signatures of vibrational coherence transfer in alkane solution. This finding holds promise towards developing a molecularly detailed understanding of the mechanism of vibrational coherence transfer processes, thanks to the ease of synthesizing derivatives based on the chemical modularity of these well studied diiron compounds. In addition to the fundamental need to characterize coherence transfer in molecular spectroscopy, we find in this set of molecules a practical utility for the nonsecular dynamics: the ability to determine the frequency of an IR-inactive mode. A coherence generated during the waiting time of the 2D-IR measurement transfers to a coherence involving the single dark CO stretching mode, which modulates some peak amplitudes in the 2D spectrum, revealing its transient excitation.

We report a spectroscopic investigation of the ultrafast dynamics of the second-generation poly(aryl ether) dendritic hydrogenase model using two-dimensional infrared (2D-IR) spectroscopy to probe the metal carbonyl vibrations of the dendrimer and a reference small molecule, [Fe(μ-S)(CO)3]2. We find that the structural dynamics of the dendrimer are reflected in a slow phase of the spectral diffusion, which is absent from [Fe(μ-S)(CO)3]2, and we relate the slow phase to the quality of the solvent for poly(aryl ether) dendrimers. We observe a solvent-dependent modulation of the initial phase of vibrational relaxation of the carbonyl groups, which we attribute to an inhibition of solvent assistance in the intramolecular vibrational redistribution process for the dendrimer. There is also a clear solvent dependence of the vibrational frequencies of both the dendrimer and [Fe(μ-S)(CO)3]2. Our data represent the first 2D-IR study of a dendritic complex and provide insight into the solvent dependence of molecular conformation in solution and the ultrafast dynamics of moderately sized, conformationally mobile compounds.

Hydrogenase enzymes enable organisms to use H2 as an energy source, having evolved extremely efficient biological catalysts for the reversible oxidation of molecular hydrogen. Small-molecule mimics of these enzymes provide both simplified models of the catalysis reactions and potential artificial catalysts that might be used to facilitate a hydrogen economy. We have studied two diiron hydrogenase mimics, μ-pdt-[Fe(CO)3]2 and μ-edt-[Fe(CO)3]2 (pdt = propanedithiolate, edt = ethanedithiolate), in a series of alkane solvents and have observed significant ultrafast spectral dynamics using two-dimensional infrared (2D-IR) spectroscopy. Since solvent fluctuations in nonpolar alkanes do not lead to substantial electrostatic modulations in a solute's vibrational mode frequencies, we attribute the spectral diffusion dynamics to intramolecular flexibility. The intramolecular origin is supported by the absence of any measurable solvent viscosity dependence, indicating that the frequency fluctuations are not coupled to the solvent motional dynamics. Quantum chemical calculations reveal a pronounced coupling between the low-frequency torsional rotation of the carbonyl ligands and the terminal CO stretching vibrations. The flexibility of the CO ligands has been proposed to play a central role in the catalytic reaction mechanism, and our results highlight that the CO ligands are highly flexible on a picosecond time scale.

In an effort to examine the role of electronic structure and oxidation states in potentially modifying intramolecular vibrational dynamics and intermolecular solvation, we have used 2D-IR to study two distinct oxidation states of an organometallic complex. The complex, [1,1'-bis(diphenylphosphino)ferrocene]tetracarbonyl chromium (DPPFCr), consists of a catalytic diphenylphosphino ferrocene redox-active component as well as a Cr that can be switched from a Cr(0) to a Cr(I) oxidation state using a chemical oxidant in dichloromethane (DCM) solution. The DPPFCr(I) radical cation is sufficiently stable to investigate with 2D-IR spectroscopy, which provides dynamical information such as vibrational relaxation, intramolecular vibrational redistribution, as well as solvation dynamics manifested as spectral diffusion. Our measurements show that the primarily intramolecular dynamical processes-vibrational relaxation and redistribution-differ significantly between the two oxidation states, with faster relaxation in the oxidized DPPFCr(I) radical cation. The primarily intermolecular spectral diffusion dynamics, however, exhibit insignificant oxidation state dependence. We speculate that the low nucleophilicity (i.e., donicity) of the DCM solvent, which is chosen to facilitate the chemical oxidation, masks any potential changes in solvation dynamics accompanying the substantial decrease in the 2.5 D molecular dipole moment of DPPFCr(I) relative to DPPFCr(0) (7.5 D).

Complementary methods of high-resolution mass spectrometry and microspectroscopy were utilized for molecular analysis of secondary organic aerosol (SOA) generated from ozonolysis of two structural monoterpene isomers: D-limonene SOA (LSOA) and α-pinene SOA (PSOA). The LSOA compounds readily formed adducts with Na(+) under electrospray ionization conditions, with only a small fraction of compounds detected in the protonated form. In contrast, a significant fraction of PSOA compounds appeared in the protonated form because of their increased molecular rigidity. Laboratory simulated aging of LSOA and PSOA, through conversion of carbonyls into imines mediated by NH3 vapors in humid air, resulted in selective browning of the LSOA sample, while the PSOA sample remained white. Comparative analysis of the reaction products in the aged LSOA and PSOA samples provided insights into chemistry relevant to formation of brown carbon chromophores. A significant fraction of carbonyl-imine conversion products with identical molecular formulas was detected in both samples. This reflects the high level of similarity in the molecular composition of these two closely related SOA materials. Several highly conjugated products were detected exclusively in the brown LSOA sample and were identified as potential chromophores responsible for the observed color change. The majority of the unique products in the aged LSOA sample with the highest number of double bonds contain two nitrogen atoms. We conclude that chromophores characteristic of the carbonyl-imine chemistry in LSOA are highly conjugated oligomers of secondary imines (Schiff bases) present at relatively low concentrations. Formation of this type of conjugated compounds in PSOA is hindered by the structural rigidity of the α-pinene oxidation products. Our results suggest that the overall light-absorbing properties of SOA may be determined by trace amounts of strong brown carbon chromophores.

Nanospray desorption electrospray ionization (nano-DESI) combined with high-resolution mass spectrometry was used for the first time for the analysis of the polar constituents of liquid petroleum crude oil samples. The analysis was performed in both positive and negative ionization modes using three solvents, one of which (acetonitrile/toluene mixture) is commonly used in petroleomics studies while two other polar solvents (acetonitrile/water and methanol/water mixtures) are generally not compatible with petroleum characterization using mass spectrometry. The results demonstrate that nano-DESI analysis efficiently ionizes petroleum constituents soluble in a particular solvent. When acetonitrile/toluene is used as a solvent, nano-DESI generates electrospray-like spectra. In contrast, strikingly different spectra were obtained using acetonitrile/water and methanol/water. Comparison with the literature data indicates that these solvents selectively extract water-soluble constituents of the crude oil. Water-soluble compounds are predominantly observed as sodium adducts in nano-DESI spectra indicating that addition of sodium to the solvent may be a viable approach for efficient ionization of water-soluble crude oil constituents. Nano-DESI enables rapid screening of different classes of compounds in crude oil samples based on their solubility in solvents that are rarely used for petroleum characterization providing better coverage of the crude oil composition as compared to electrospray ionization (ESI). It also enables rapid characterization of water-soluble components of petroleum samples that is difficult to perform using traditional approaches.

Reactive nanospray desorption electrospray ionization (nano-DESI) combined with high-resolution mass spectrometry was utilized for the analysis of secondary organic aerosol produced through ozonolysis of limonene (LSOA). Previous studies have shown that LSOA constituents are multifunctional compounds containing at least one aldehyde or ketone groups. In this study, we used the selectivity of the Girard's reagent T (GT) toward carbonyl compounds to examine the utility of reactive nano-DESI for the analysis of complex organic mixtures. In these experiments, 1-100 μM GT solutions were used as the working solvents for reactive nano-DESI analysis. Abundant products from the single addition of GT to LSOA constituents were observed at GT concentrations in excess of 10 μM. We found that LSOA dimeric and trimeric compounds react with GT through a simple addition reaction resulting in formation of the carbinolamine derivative. In contrast, reactions of GT with monomeric species result in the formation of both the carbinolamine and the hydrazone derivatives. In addition, several monomers did not react with GT on the time scale of our experiment. These molecules were characterized by relatively high values of the double bond equivalent and low oxygen content. Furthermore, because addition of a charged GT tag to a neutral molecule eliminates the discrimination against the low proton affinity compounds in the ionization process, reactive nano-DESI analysis enables quantification of individual compounds in the complex mixture. For example, we were able to estimate for the first time the amounts of dimers and trimers in the LSOA mixture. Specifically, we found that the most abundant LSOA dimer was detected at the ~0.5 pg level and the total amount of dimers and trimers in the analyzed sample was ~11 pg. Our results indicate that reactive nano-DESI is a valuable approach for examining the presence of specific functional groups and for the quantification of compounds possessing these groups in complex mixtures.