Light-driven chloride transport kinetics of halorhodopsin

Despite growing interest in light-driven ion pumps for use in optogenetics, current estimates of their transport rates span two orders of magnitude due to challenges in measuring slow transport processes and determining protein concentration and/or orientation in membranes in vitro. In this study, we report, to our knowledge, the first direct quantitative measurement of light-driven Cl transport rates of the anion pump halorohodopsin from Natronomonas pharaonis (NpHR). We used light-interfaced voltage clamp measurements on NpHR-expressing oocytes to obtain a transport rate of 219 (± 98) Cl /protein/s for a photon flux of 630 photons/protein/s. The measurement is consistent with the literature-reported quantum efficiency of ∼30% for NpHR, i.e., 0.3 isomerizations per photon absorbed. To reconcile our measurements with an earlier-reported 20 ms rate-limiting step, or 35 turnovers/protein/s, we conducted, to our knowledge, novel consecutive single-turnover flash experiments that demonstrate that under continuous illumination, NpHR bypasses this step in the photocycle

Heterologous Assembly of Pleomorphic Bacterial Microcompartment Shell Architectures Spanning the Nano‐ to Microscale

Many bacteria use protein-based organelles known as bacterial microcompartments (BMCs) to organize and sequester sequential enzymatic reactions. Regardless of their specialized metabolic function, all BMCs are delimited by a shell made of multiple structurally redundant, yet functionally diverse, hexameric (BMC-H), pseudohexameric/trimeric (BMC-T), or pentameric (BMC-P) shell protein paralogs. When expressed without their native cargo, shell proteins have been shown to self-assemble into 2D sheets, open-ended nanotubes, and closed shells of ≈40 nm diameter that are being developed as scaffolds and nanocontainers for applications in biotechnology. Here, by leveraging a strategy for affinity-based purification, it is demonstrated that a wide range of empty synthetic shells, many differing in end-cap structures, can be derived from a glycyl radical enzyme-associated microcompartment. The range of pleomorphic shells observed, which span ≈2 orders of magnitude in size from ≈25 nm to ≈1.8 µm, reveal the remarkable plasticity of BMC-based biomaterials. In addition, new capped nanotube and nanocone morphologies are observed that are consistent with a multicomponent geometric model in which architectural principles are shared among asymmetric carbon, viral protein, and BMC-based structures

Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme.

Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling bacterial microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about -210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins

Functionalization of Bacterial Microcompartment Shell Proteins With Covalently Attached Heme

Heme is a versatile redox cofactor that has considerable potential for synthetic biology and bioelectronic applications. The capacity to functionalize non-heme-binding proteins with covalently bound heme moieties in vivo could expand the variety of bioelectronic materials, particularly if hemes could be attached at defined locations so as to facilitate position-sensitive processes like electron transfer. In this study, we utilized the cytochrome maturation system I to develop a simple approach that enables incorporation of hemes into the backbone of target proteins in vivo. We tested our methodology by targeting the self-assembling bacterial microcompartment shell proteins, and inserting functional hemes at multiple locations in the protein backbone. We found substitution of three amino acids on the target proteins promoted heme attachment with high occupancy. Spectroscopic measurements suggested these modified proteins covalently bind low-spin hemes, with relative low redox midpoint potentials (about -210 mV vs. SHE). Heme-modified shell proteins partially retained their self-assembly properties, including the capacity to hexamerize, and form inter-hexamer attachments. Heme-bound shell proteins demonstrated the capacity to integrate into higher-order shell assemblies, however, the structural features of these macromolecular complexes was sometimes altered. Altogether, we report a versatile strategy for generating electron-conductive cytochromes from structurally-defined proteins, and provide design considerations on how heme incorporation may interface with native assembly properties in engineered proteins

Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate.

Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don't compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a "wiffleball" architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology

Thermodynamics of the Electron Acceptors in <i>Heliobacterium modesticaldum</i>: An Exemplar of an Early Homodimeric Type I Photosynthetic Reaction Center

The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigment–protein complex capable of conducting (bacterio)­chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S]^2+/1+ cluster (F_X) in reaction centers from <i>Heliobacterium modesticaldum</i>. The F_X cluster was titrated chemically and monitored by (i) the decrease in the level of stable P₈₀₀ photobleaching by optical spectroscopy, (ii) the loss of the light-induced <i>g</i> ≈ 2 radical from P₈₀₀^+• following a single-turnover flash, (iii) the increase in the low-field resonance at 140 mT attributed to the <i>S</i> = ³/₂ ground spin state of F_X^–, and (iv) the loss of the spin-correlated P₈₀₀⁺ F_X^– radical pair following a single-turnover flash. These four techniques led to similar estimations of the midpoint potential for F_X of −502 ± 3 mV (<i>n</i> = 0.99), −496 ± 2 mV (<i>n</i> = 0.99), −517 ± 10 mV (<i>n</i> = 0.65), and −501 ± 4 mV (<i>n</i> = 0.84), respectively, with a consensus value of −504 ± 10 mV (converging to <i>n</i> = 1). Under conditions in which F_X is reduced, the long-lived (∼15 ms) P₈₀₀⁺ F_X^– state is replaced by a rapidly recombining (∼15 ns) P₈₀₀⁺A₀^– state, as shown by ultrafast optical experiments. There was no evidence of the presence of a P₈₀₀⁺ A₁^– spin-correlated radical pair by electron paramagnetic resonance (EPR) under these conditions. The midpoint potentials of the two [4Fe-4S]^2+/1+ clusters in the low-molecular mass ferredoxins were found to be −480 ± 11 mV/–524 ± 13 mV for PshBI, −453 ± 6 mV/–527 ± 6 mV for PshBII, and −452 ± 5 mV/–533 ± 8 mV for HM1_2505 as determined by EPR spectroscopy. F_X is therefore suitably poised to reduce one [4Fe-4S]^2+/1+ cluster in these mobile electron carriers. Using the measured midpoint potential of F_X and a quasi-equilibrium model of charge recombination, the midpoint potential of A₀ was estimated to be −854 mV at room temperature. The midpoint potentials of A₀ and F_X are therefore 150–200 mV less reducing than their respective counterparts in Photosystem I of cyanobacteria and plants. This places the redox potential of the F_X cluster in heliobacteria approximately equipotential to the highest-potential iron–sulfur cluster (F_A) in Photosystem I, consistent with its assignment as the terminal electron acceptor