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At least 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Biophysical and Biochemical Characterization of TP0037, a D-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum

A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism’s robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum. Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein’s propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum’s d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Improved accuracy and transferability of molecular-orbital-based machine learning: Organics, transition-metal complexes, non-covalent interactions, and transition states

We report molecular-orbital-based machine learning (MOB-ML) provides a general framework for the prediction of accurate correlation energies at the cost of obtaining molecular orbitals. The application of Nesbet’s theorem makes it possible to recast a typical extrapolation task, training on correlation energies for small molecules and predicting correlation energies for large molecules, into an interpolation task based on the properties of orbital pairs. We demonstrate the importance of preserving physical constraints, including invariance conditions and size consistency, when generating the input for the machine learning model. Numerical improvements are demonstrated for different datasets covering total and relative energies for thermally accessible organic and transition-metal containing molecules, non-covalent interactions, and transition-state energies. MOB-ML requires training data from only 1% of the QM7b-T dataset (i.e., only 70 organic molecules with seven and fewer heavy atoms) to predict the total energy of the remaining 99% of this dataset with sub-kcal/mol accuracy. This MOB-ML model is significantly more accurate than other methods when transferred to a dataset comprising of 13 heavy atom molecules, exhibiting no loss of accuracy on a size intensive (i.e., per-electron) basis. It is shown that MOB-ML also works well for extrapolating to transition-state structures, predicting the barrier region for malonaldehyde intramolecular proton-transfer to within 0.35 kcal/mol when only trained on reactant/product-like structures. Finally, the use of the Gaussian process variance enables an active learning strategy for extending the MOB-ML model to new regions of chemical space with minimal effort. We demonstrate this active learning strategy by extending a QM7b-T model to describe non-covalent interactions in the protein backbone–backbone interaction dataset to an accuracy of 0.28 kcal/mol.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Crystal structure of the Arabidopsis SPIRAL2 C-terminal domain reveals a p80-Katanin-like domain

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

De novo design of drug-binding proteins with predictable binding energy and specificity

The de novo design of small molecule–binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase–1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule–binding proteins with tuned interaction energies is feasible entirely from computation.

Science & Technology - Other Topics↗
DOE OSTI2023

Evaluating the active site-substrate interplay between x-ray crystal structure and molecular dynamics in chorismate mutase

Designing realistic quantum mechanical (QM) models of enzymes is dependent on reliably discerning and modeling residues, solvents, and cofactors important in crafting the active site microenvironment. Interatomic van der Waals contacts have previously demonstrated usefulness toward designing QM-models, but their measured values (and subsequent residue importance rankings) are expected to be influenceable by subtle changes in protein structure. Using chorismate mutase as a case study, this work examines the differences in ligand-residue interatomic contacts between an x-ray crystal structure and structures from a molecular dynamics simulation. Select structures are further analyzed using symmetry adapted perturbation theory to compute ab initio ligand-residue interaction energies. The findings of this study show that ligand-residue interatomic contacts measured for an x-ray crystal structure are not predictive of active site contacts from a sampling of molecular dynamics frames. In addition, the variability in interatomic contacts among structures is not correlated with variability in interaction energies. However, the results spotlight using interaction energies to characterize and rank residue importance in future computational enzymology workflows.

Chemistry↗
DOE OSTI2021

Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Thermodynamic Basis for the Stabilization of Helical Peptoids by Chiral Sidechains

Peptoids are a class of highly customizable biomimetic foldamers that retain properties from both proteins and polymers. It has been shown that peptoids can adopt peptide-like secondary structures through the careful selection of sidechain chemistries, but the underlying conformational landscapes that drive these assemblies at the molecular level remain poorly understood. Given the high flexibility of the peptoid backbone, it is essential that methods applied to study peptoid secondary structure formation possess the requisite sensitivity to discriminate between structurally similar yet energetically distinct microstates. In this work, a generalizable simulation scheme is used to robustly sample the complex folding landscape of various 12mer polypeptoids, resulting in a predictive model that links sidechain chemistry with preferential assembly into one of 12 accessible backbone motifs. Using a variant of the metadynamics sampling method, four peptoid dodecamers are simulated in water: sarcosine, N-(1-phenylmethyl)glycine (Npm), (S)-N-(1-phenylethyl)glycine (Nspe), and (R)-N-(1-phenylethyl)glycine (Nrpe)–to determine the underlying entropic and energetic impacts of hydrophobic and chiral peptoid sidechains on secondary structure formation. Our results indicate that the driving forces to assemble Nrpe and Nspe sequences into polyproline type-I helices in water are found to be enthalpically driven, with small benefits from an entropic gain for isomerization and steric strain due to the presence of the chiral center. The minor entropic gains from bulky chiral sidechains in Nrpe- and Nspe-containing peptoids can be explained through increased configurational entropy in the cis state. However, overall assembly into a helix is found to be overall entropically unfavorable. Furthermore, these results highlight the importance of considering the many various competing interactions in the rational design of peptoid secondary structure building blocks.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Dissociative electron attachment to amide bond containing molecules: N-ethylformamide and N-ethylacetamide

To advance our quest to understand the role of low energy electrons in biomolecular systems, we performed investigations on dissociative electron attachment (DEA) to gas-phase N-ethylformamide (NEF) and N-ethylacetamide (NEA) molecules. Both molecules contain the amide bond, which is the linkage between two consecutive amino acid residues in proteins. Thus, their electron-induced dissociation can imitate the resonant behavior of the DEA process in more complex biostructures. Our experimental results indicate that in these two molecules, the dissociation of the amide bond results in a double resonant structure with peaks at ~5 eV and 9 eV. We also determined the energy position of resonant states for several negative ions, i.e., the other dissociation products from NEF and NEA. Our predictions of dissociation channels were supported by density functional theory calculations of the corresponding threshold energies. Our results and those previously reported for small amides and peptides imply the fundamental nature for breakage of the amide bond through the DEA process.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

An atypical BRCT–BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Assessment of software methods for estimating protein-protein relative binding affinities

A growing number of computational tools have been developed to accurately and rapidly predict the impact of amino acid mutations on protein-protein relative binding affinities. Such tools have many applications, for example, designing new drugs and studying evolutionary mechanisms. In the search for accuracy, many of these methods employ expensive yet rigorous molecular dynamics simulations. By contrast, non-rigorous methods use less exhaustive statistical mechanics, allowing for more efficient calculations. However, it is unclear if such methods retain enough accuracy to replace rigorous methods in binding affinity calculations. This trade-off between accuracy and computational expense makes it difficult to determine the best method for a particular system or study. Here, eight non-rigorous computational methods were assessed using eight antibody-antigen and eight non-antibody-antigen complexes for their ability to accurately predict relative binding affinities (ΔΔG) for 654 single mutations. In addition to assessing accuracy, we analyzed the CPU cost and performance for each method using a variety of physico-chemical structural features. This allowed us to posit scenarios in which each method may be best utilized. Most methods performed worse when applied to antibody-antigen complexes compared to non-antibody-antigen complexes. Rosetta-based JayZ and EasyE methods classified mutations as destabilizing (ΔΔG < -0.5 kcal/mol) with high (83–98%) accuracy and a relatively low computational cost for non-antibody-antigen complexes. Some of the most accurate results for antibody-antigen systems came from combining molecular dynamics with FoldX with a correlation coefficient (r) of 0.46, but this was also the most computationally expensive method. Overall, our results suggest these methods can be used to quickly and accurately predict stabilizing versus destabilizing mutations but are less accurate at predicting actual binding affinities. This study highlights the need for continued development of reliable, accessible, and reproducible methods for predicting binding affinities in antibody-antigen proteins and provides a recipe for using current methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural Insights into Mechanisms Underlying Mitochondrial and Bacterial Cytochrome c Synthases

Mitochondrial holocytochrome c synthase (HCCS) is an essential protein in assembling cytochrome c (cyt c) of the electron transport system. HCCS binds heme and covalently attaches the two vinyls of heme to two cysteine thiols of the cyt c CXXCH motif. Human HCCS recognizes both cyt c and cytochrome c1 of complex III (cytochrome bc1). HCCS is mutated in some human diseases and it has been investigated recombinantly by mutational, biochemical, and reconstitution studies in the past decade. Here, we employ structural prediction programs (e.g., AlphaFold 3) on HCCS and its two substrates, heme and cytochrome c. The results, when combined with spectroscopic and functional analyses of HCCS and variants, provide insights into the structural basis for heme binding, apocyt c binding, covalent attachment, and release of the holocyt c product. Results from in vitro reconstitution of purified human HCCS using cyt c and cyt c1 peptides as acceptors are consistent with the structural modeling of substrate binding. Reconstitution of HCCS and cyt c1 provides an approach to studying cyt c1 assembly, which has been refractile to recombinant in vivo reconstitution (unlike HCCS and cyt c). We propose a structural basis for release of the holocyt c product from HCCS based on in vitro studies and on cryoEM structures of the bacterial cyt c synthase (CcsBA) active site. We analyze the kinetoplastid mitochondrial synthase (KCCS), and hypothesize a molecular evolutionary path from mitochondrial endosymbiosis to the current HCCS.

Biochemistry & Molecular Biology↗
DOE OSTI2024

Borg extrachromosomal elements of methane-oxidizing archaea have conserved and expressed genetic repertoires

Borgs are huge extrachromosomal elements (ECE) of anaerobic methane-consuming “Candidatus Methanoperedens” archaea. Here, we used nanopore sequencing to validate published complete genomes curated from short reads and to reconstruct new genomes. 13 complete and four near-complete linear genomes share 40 genes that define a largely syntenous genome backbone. We use these conserved genes to identify new Borgs from peatland soil and to delineate Borg phylogeny, revealing two major clades. Remarkably, Borg genes encoding nanowire-like electron-transferring cytochromes and cell surface proteins are more highly expressed than those of host Methanoperedens, indicating that Borgs augment the Methanoperedens activity in situ. We reconstructed the first complete 4.00 Mbp genome for a Methanoperedens that is inferred to be a Borg host and predicted its methylation motifs, which differ from pervasive TC and CC methylation motifs of the Borgs. Thus, methylation may enable Methanoperedens to distinguish their genomes from those of Borgs. Very high Borg to Methanoperedens ratios and structural predictions suggest that Borgs may be capable of encapsulation. The findings clearly define Borgs as a distinct class of ECE with shared genomic signatures, establish their diversification from a common ancestor with genetic inheritance, and raise the possibility of periodic existence outside of host cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Uncovering Sequence and Structural Characteristics of Fungal Expansin‐Related Proteins With Potential to Drive Substrate Targeting

Expansins loosen plant cell wall networks through disrupting non-covalent bonds between cellulose microfibrils and matrix polysaccharides. Whereas expansins were first discovered in plants, expansin-related proteins have since been identified in bacteria and fungi. The biological function of microbial expansins remains unclear; however, several studies have shown distinct binding preferences toward different structural polysaccharides. Earlier studies of bacterial expansin-related proteins uncovered sequence and structural features that correlate to substrate binding. Herein, 20 fungal expansin-related sequences were recombinantly produced in Komagataella phaffii, and the purified proteins were compared in terms of substrate binding to cellulosic and chitinous substrates. The impact of pH on the zeta potential of prioritized substrates was also measured, and Principal Component Analysis was performed to uncover correlations between protein characteristics (e.g., pI, hydrophobicity, surface charge distribution) and measured substrate binding preferences. Whereas acidic proteins with a predicted pI less than 5.0 preferentially bound to chitin, basic proteins with pI greater than 8.0 preferentially bound to xylan and xylan-containing fiber. Similar to many cellulases, binding to cellulose was correlated to relatively high aromatic amino acid content in the protein sequence and presence of a carbohydrate binding module (CBM), which in the case of expansins is a C-terminal CBM63. Whereas overall sequence characteristics could be correlated to substrate binding preference, the identity of amino acids occupying conserved positions that impact protein activity was better correlated with loosenin versus expansin classifications.

chitin↗
DOE OSTI2023

Hydrodynamics of thermally driven chiral suspensions

Considerable effort has been directed towards the characterization of chiral mesoscale structures, as shown in chiral protein assemblies and carbon nanotubes. Here, we establish a thermally driven hydrodynamic description for the actuation and separation of mesoscale chiral structures in a fluid medium. Cross-flow of a Newtonian liquid with a thermal gradient gives rise to an effective torque that propels each particle of a chiral suspension according to its handedness. In turn, the chiral suspension alters the liquid flow, which thus acquires a transverse (chiral) velocity component. Since observation of the predicted effects requires a low degree of sophistication, our work provides an efficient and inexpensive approach to test and calibrate chiral particle propulsion and separation strategies.

Mechanics↗
DOE OSTI2021

The genotype-phenotype landscape of an allosteric protein

Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. Here, we report the large-scale measurement of the genotype-phenotype landscape for an allosteric protein: the lac repressor from Escherichia coli , LacI. Using a method that combines long-read and short-read DNA sequencing, we quantitatively measure the dose-response curves for nearly 10 5 variants of the LacI genetic sensor. The resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to discover a new band-stop phenotype that challenges conventional models of allostery and that emerges from combinations of nearly silent amino acid substitutions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Rational Design of Novel Biomimetic Sequence-Defined Polymers for Mineralization Applications

Silica biomineralization is a naturally occurring process, wherein organisms use proteins and other biological structures to direct the formation of complex, hierarchical nanostructures. Discovery and characterization of such proteins and their underlying mechanisms spurred significant efforts to identify routes for biomimetic mineralization that reproduce the exquisite shapes and size selectivities found in nature. A common strategy has been the use of short peptide sequences with chemistry mimicking those found in natural systems, such as the use of the silaffin-derived R5 peptide. While progress has been made using this approach, there are many limitations that have prevented breakthroughs in biomimicry. To advance our ability to use charged macromolecules for silica formation, we propose to use sequence-defined synthetic polymers known as peptoids, or N-substituted polyglycines, which present significant capability for the precise tuning of sequence and structure beyond what can often be achieved with peptides alone. This study presents a computationally predicted design of these polymers that leads to the controlled formation of silica nanomaterials. We investigate surface adsorption and the mineralization process through analysis of binding mechanisms and energetics of the R5 system. Next, we synthesized two R5-inspired peptoids and validated our prediction in the design of mineralization polymers through characterization using surface plasmon resonance and electron microscopy. Here, this computationally guided study holds great promise for designing new sequences with unprecedented control of the placement of chemical functional groups, thus allowing for further unraveling of silicification mechanisms and the eventual design of sequence-defined synthetic polymers leading to the predictive synthesis of nanostructured functional materials.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Mechanism and dynamics of fatty acid photodecarboxylase

Photoenzymes are rare biocatalysts driven by absorption of a photon at each catalytic cycle; they inspire development of artificial photoenzymes with valuable activities. Fatty acid photodecarboxylase (FAP) is a natural photoenzyme that has potential applications in the bio-based production of hydrocarbons, yet its mechanism is far from fully understood. RATIONALE To elucidate the mechanism of FAP, we studied the wild-type (WT) enzyme from Chlorella variabilis (CvFAP) and variants with altered active-site residues using a wealth of techniques, including static and time-resolved crystallography and spectroscopy, as well as biochemical and computational approaches. RESULTS A 1.8-Å-resolution CvFAP x-ray crystal structure revealed a dense hydrogen-bonding network positioning the fatty acid carboxyl group in the vicinity of the flavin adenine dinucleotide (FAD) cofactor. Structures solved from free electron laser and low-dose synchrotron x-ray crystal data further highlighted an unusual bent shape of the oxidized flavin chromophore, and showed that the bending angle (14°) did not change upon photon absorption (step 1) or throughout the photocycle. Calculations showed that bending substantially affected the energy levels of the flavin. Structural and spectroscopic analysis of WT and mutant proteins targeting two conserved active-site residues, R451 and C432, demonstrated that both residues were crucial for proper positioning of the substrate and water molecules and for oxidation of the fatty acid carboxylate by 1 FAD* (~300 ps in WT FAP) to form FAD ∙– (step 2). Time-resolved infrared spectroscopy demonstrated that decarboxylation occured quasi-instantaneously upon this forward electron transfer, consistent with barrierless bond cleavage predicted by quantum chemistry calculations and with snapshots obtained by time-resolved crystallography. Transient absorption spectroscopy in H 2 O and D 2 O buffers indicated that back electron transfer from FAD ∙– was coupled to and limited by transfer of an exchangeable proton or hydrogen atom (step 3). Unexpectedly, concomitant with FAD ∙– reoxidation (to a red-shifted form FAD RS ) in 100 ns, most of the CO 2 product was converted, most likely into bicarbonate (as inferred from FTIR spectra of the cryotrapped FAD RS intermediate). Calculations indicated that this catalytic transformation involved an active-site water molecule. Cryo-Fourier transform infrared spectroscopy studies suggested that bicarbonate formation (step 4) was preceded by deprotonation of an arginine residue (step 3). At room temperature, the remaining CO 2 left the protein in 1.5 μs (step 4'). The observation of residual electron density close to C432 in electron density maps derived from time-resolved and cryocrystallography data suggests that this residue may play a role in stabilizing CO 2 and/or bicarbonate. Three routes for alkane formation were identified by quantum chemistry calculations; the one shown in the figure is favored by the ensemble of experimental data. CONCLUSION Finally, we provide a detailed and comprehensive characterization of light-driven hydrocarbon formation by FAP, which uses a remarkably complex mechanism including unique catalytic steps. We anticipate that our results will help to expand the green chemistry toolkit.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗