David M. Tiede

Valence dynamics of transition metals are central to oxygen evolution reaction (OER) mechanisms, yet in situ spectroscopic studies capturing these dynamics within amorphous catalysts remain limited. Here, we present in situ X-ray spectroscopy of amorphous catalysts, where CoBi exhibits higher OER performance compared to CoPi. While our data shows successive valence changes toward high-valent states in both catalysts, distinct initial electro-structural states dictate the intermediate evolution. Co(III) dominates in both catalysts at open-circuit potential, yet the Co(III)–O–Co(III) bridge structure observed in CoBi is absent in CoPi. Under active OER, Co(IV) species are identified as distinguishable spectral intensities; these are assigned to highly hybridized low-spin (LS) Co(IV) with reduced electron–electron repulsion, as validated by resonant inelastic X-ray spectroscopy (RIXS) analysis of Li0.75CoO2. Density functional theory supports the conversion from tetrahedral (Td) Co(II) to octahedral (Oh) Co(III) and suggests that factors other than thermodynamic pathways are likely responsible for the performance gap. The lower formal oxidation, larger Co–O length, smaller Co–O–Co angle, prominent pre-edge dipole character and absent oxo-bridging in CoPi suggest a kinetically inefficient Co(IV) state. These findings demonstrate that the initial electro-structure strongly influences the formation of productive high-valent active species and the resulting OER activity.

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here, we describe the development of an efficient and rapid method for the in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.

Bacterial microcompartments (BMCs) are self-assembling protein shell structures that are widely investigated across a broad range of biological and abiotic chemistry applications. A central challenge in BMC research is the targeted capture of enzymes during shell assembly. While crystallography and cryo-EM techniques have been successful in determining BMC shell structures, there has been only limited success in visualizing the location of BMC-captured enzyme cargo. Here, we demonstrate the opportunity to use small-angle X-ray scattering (SAXS) and pair distance distribution function (PDDF) measurements combined with quantitative comparison to coordinate structure models as an approach to characterize BMC shell structures in solution conditions directly relevant to biochemical function. Using this approach, we analyzed BMC shells from Haliangium ochraceum (HO) that were isolated following expression in E. coli. The analysis allowed the BMC shell structures and the extent of encapsulated enzyme cargo to be identified. Notably, the results demonstrate that HO-BMC shells adventitiously capture significant amounts of cytoplasmic cargo during assembly in E. coli. Our findings highlight the utility of SAXS/PDDF analysis for evaluating BMC architectures and enzyme encapsulation, offering valuable insights for designing BMC shells as platforms for biological and abiotic catalyst capture within confined environments.

Directed assembly of abiotic catalysts onto biological redox protein frameworks is of interest as an approach for the synthesis of biohybrid catalysts that combine features of both synthetic and biological materials. In this report, we provide a multiscale characterization of the platinum nanoparticle (NP) hydrogen-evolving catalysts that are assembled by light-driven reductive precipitation of platinum from an aqueous salt solution onto the photosystem I protein (PSI), isolated from cyanobacteria as trimeric PSI. The resulting PSI-NP assemblies were analyzed using a combination of X-ray energy-dispersive spectroscopy (XEDS), high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), small-angle X-ray scattering (SAXS), and high-energy X-ray scattering with atomic pair distribution function (PDF) analyses. The results show that the PSI-supported NPs are approximately 1.8 nm diameter disk-shaped particles that assemble at discrete sites with 145 Å separation. This separation is too large to be consistent with NP nucleation and growth at a site adjacent to the F_B cofactor site. Instead, we suggest a mechanism for NP growth at hydrophobic sites on the PSI stromal surface. The NPs photoreductively assembled on the PSI stromal surface are found to be analogous to the nanostructures produced by successive cycles of atomic layer deposition (ALD) of platinum onto 40 nm porous anodic alumina oxide supports, although the mechanisms for nucleation appear to differ. This work establishes a foundation for the investigation of the reductive assembly of abiotic metal catalysts at sites connected to photochemically reducing equivalent production in PSI.

We investigate the ultrafast electronic and thermal properties of the bulk amorphous cobalt oxide water oxidation catalysts cobalt-phosphate (CoPi) and cobalt borate (CoBi) using optical pump/X-ray probe correlated with in situ electrochemical transient absorption spectroscopy. The electronic signature of a light-generated intermediate species is compared to steady-state in situ heating X-ray absorption spectroscopy (XAS), suggestive of non-thermal contributions to charge transfer formation on ultrafast timescales. With Co K-edge transient absorption spectroscopy, we observe a net photoreduction following 400 nm excitation that also initiates a potential-dependent increase in the excited-state fraction of trapped charge carriers that persists on nanosecond to microsecond timescales and is identified as a potential precursor state to oxygen evolving reactions (OER). Distinctly, the formation of excited-state species cannot be fully explained by photothermal reaction dynamics alone, with potential contributions from electronic motion acting in concert with macroscopic redox activity. The results can influence the design of water-splitting catalytic materials for tuning dimensionality, confinement, and charge delocalization across structures.

Bacterial microcompartments (BMCs) are self-assembling, protein shell structures that are widely investigated across a broad range of biological and abiotic chemistry applications. A central challenge in BMC research is the targeted capture of enzymes during shell assembly. While crystallography and cryo-EM techniques have been successful in determining BMC shell structures, there has been only limited success in visualizing the location of BMC-captured enzyme cargo. Here, we demonstrate the opportunity to use small angle X-ray scattering (SAXS) and pair density distribution function (PDDF) measurements combined with quantitative comparison to coordinate structure models as an approach to characterize BMC shell structures in solution conditions directly relevant to biochemical function. Using this approach, we analyzed BMC shells from Haliangium ochraceum that were isolated following expression in E. coli . The analysis allowed BMC shell structures and the extent of encapsulated enzyme cargo to be identified. Notably, the results demonstrate that HO-BMC shells adventitiously capture significant amounts of cytoplasmic cargo during assembly in E. coli . Our findings highlight the utility of SAXS/PDDF analysis for evaluating BMC architectures and enzyme encapsulation, offering valuable insights for designing BMC shells as platforms for biological and abiotic catalyst capture within confined environments.

Bacterial microcompartments (BMCs) are proteinaceous organelles that self-assemble into selectively permeable shells that encapsulate enzymatic cargo. BMCs enhance catalytic pathways by reducing crosstalk among metabolites, preventing harmful intermediates from leaking into the cytosol, and increasing reaction efficiency via enzyme colocalization. The intrinsic properties of BMCs make them attractive for biotechnological engineering. However, in vivo expression methods for shell synthesis have significant drawbacks that limit the potential design space for these nanocompartments. Here we describe the development of a new, efficient, and rapid method for in vitro assembly of BMC shells from their protein building blocks. Our method enables large-scale construction of BMC shells by utilizing urea as a chaotropic agent to control self-assembly, and provides an approach for encapsulation of both biotic and abiotic cargo under a broad range of reaction conditions. We demonstrate an enhanced level of control over the assembly of BMC shells in vitro and expand the design parameter space for engineering BMC systems with specialized and enhanced catalytic properties.

The synthesis and investigation of [Rh(DHMPE)(2)][BF4] (1) are reported. 1 features proton-responsive 1,2-bis[(dihydroxymethyl)phosphino]ethane (DHMPE) ligands, which readily capture CO2 from atmospheric sources upon deprotonation. The protonation state of the DHMPE ligand was observed to have a significant impact on the catalytic reactivity of 1 with CO2. Deprotonation and CO2 binding to 1 result in a similar to 10-fold rate enhancement in catalytic degenerate CO2 reduction with formate, monitored by C-12/C-13 isotope exchange between (HCO2-)-C-12 and (CO2)-C-13. Studies performed using a similar complex lacking the hydroxyl ligand functionality ([Rh(DEPE)(2)][BF4] where DEPE = 1,2-bis(diethylphosphino)ethane) do not show the same rate enhancements when base is added. Based upon the cation-dependent activity of the catalyst, Eyring analysis, and cation sequestration experiments, CO2 binding to 1 is proposed to facilitate preorganization of formate/CO2 in the transition state via ligand-based encapsulation of Na+ or K+ cations to lower the activation energy and increase the observed catalytic rate. Incorporation of proton-responsive DHMPE ligands provides a unique approach to accelerate the kinetics of catalytic CO2 reduction to formate.

X-ray scattering has become a major tool in the structural characterization of nanoscale materials. Thanks to the widely available experimental and computational atomic models, coordinate-based X-ray scattering simulation has played a crucial role in data interpretation in the past two decades. However, simulation of real-space pair distance distribution functions (PDDFs) from small- and wide-angle X-ray scattering, SAXS/WAXS, has been relatively less exploited. This study presents a comparison of PDDF simulation methods, which are applied to molecular structures that range in size from β-cyclodextrin [1 kDa molecular weight (MW), 66 non-hydrogen atoms] to the satellite tobacco mosaic virus capsid (1.1 MDa MW, 81 960 non-hydrogen atoms). The results demonstrate the power of interpretation of experimental SAXS/WAXS from the real-space view, particularly by providing a more intuitive method for understanding of partial structure contributions. Furthermore, the computational efficiency of PDDF simulation algorithms makes them attractive as approaches for the analysis of large nanoscale materials and biological assemblies. The simulation methods demonstrated in this article have been implemented in stand-alone software, SolX 3.0, which is available to download from https://12idb.xray.aps.anl.gov/solx.html.