Hideaki Ogata

Protein‐based supramolecules require precise arrangement of building blocks. A regular‐triangle trimer (cp‐c555)3 has been constructed using an α‐helix‐inserted‐circular permutant (cp‐c555) of Aquifex aeolicus cytochrome (cyt) c555, where the trimers may dissociate to monomers. In this study, we stabilized the regular‐triangle structure by constructing a cyclic regular‐triangle of three α‐helix‐linked cyt c555 molecules using sortase‐mediated ligation (SML). Comparing SML using sortase A for six cp‐c555 variant trimers, the variant with GGG at the N‐terminus and LPETG at the C‐terminus reacted most efficiently. OP‐(c555)3 was designed, in which two cyt c555 molecules were fused using an α‐helix. The cyt c555 C‐terminal region was attached to the N‐terminus of the dimer and the cyt c555 N‐terminal region was attached to the C‐terminus of the dimer using the same α‐helix. OP‐(c555)3 was expressed in Escherichia coli cells and the termini were connected by SML, forming a cyclic regular‐triangle, CL‐(c555)3. CL‐(c555)3 showed higher thermostability than (cp‐c555)3 and OP‐(c555)3. CL‐(c555)3 structural stability was confirmed using high speed‐atomic force microscopy. The X‐ray crystal structure of CL‐(c555)3 showed a cyclic structure and a nanoporous supramolecular assembly. These results demonstrate that a nanoporous supramolecular assembly can be constructed by designing a cyclic molecule with a central hole using SML.

Overexpression of antibody light chains in small plasma cell clones can lead to misfolding and aggregation. On the other hand, the formation of amyloid fibrils from antibody light chains is related to amyloidosis. Although aggregation of antibody light chain is an important issue, atomic-level structural examinations of antibody light chain aggregates are sparse. In this study, we present an antibody light chain that maintains an equilibrium between its monomeric and tetrameric states. According to data from X-ray crystallography, thermodynamic and kinetic measurements, as well as theoretical studies, this antibody light chain engages in 3D domain swapping within its variable region. Here, a pair of domain-swapped dimers creates a tetramer through hydrophobic interactions, facilitating the revelation of the domain-swapped structure. The negative cotton effect linked to the β-sheet structure, observed around 215 nm in the circular dichroism (CD) spectrum of the tetrameric variable region, is more pronounced than that of the monomer. This suggests that the monomer contains less β-sheet structures and exhibits greater flexibility than the tetramer in solution. These findings not only clarify the domain-swapped structure of the antibody light chain but also contribute to controlling antibody quality and advancing the development of future molecular recognition agents and drugs.

We report the first neutron structure of [NiFe]-hydrogenase in its oxidized state. This study leads to new insights into the oxidized active site and visualization of the protons characteristic of the oxidized enzyme.

Formate dehydrogenases (FDHs) catalyze the two-electron oxidation of formate to carbon dioxide. FDHs can be divided into several groups depending on their subunit composition and active-site metal ions. Metal-dependent (Mo- or W-containing) FDHs from prokaryotic organisms belong to the superfamily of molybdenum enzymes and are members of the dimethylsulfoxide reductase family. In this short review, recent progress in the structural analysis of FDHs together with their potential biotechnological applications are summarized.

Site-directed mutagenesis of the sensory [FeFe] hydrogenase from Thermotoga maritima reveals new insight into how the protein environment tunes the active site properties for its sensory role.

Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers connected via a [4Fe–4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron–sulfur cluster domain: a ‘closed bridge’ and an ‘open bridge’ conformation, where a Zn²⁺ site may act as a ‘hinge’ allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD⁺ reduction/NADH oxidation site.

Hydrogenases catalyze the reversible oxidation of H₂. Carbon monoxide (CO) is known to be a competitive inhibitor of O₂-sensitive [NiFe]-hydrogenases. Although the activities of some O₂-tolerant [NiFe]-hydrogenases are unaffected by CO, the partially O₂-tolerant [NiFe]-hydrogenase from <italic>Citrobacter</italic> sp. S-77 (S77-HYB) is inhibited by CO. In this work, the CO-bound state of S77-HYB was characterized by activity assays, spectroscopic techniques and X-ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni²⁺ state, and Fourier-transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni–Fe active site of S77-HYB. These results suggest a positive correlation between O₂ and CO tolerance in [NiFe]-hydrogenases.

Sulfur isotope fractionation resulting from microbial sulfate reduction (MSR) provides some of the earliest evidence of life, and secular variations in fractionation values reflect changes in biogeochemical cycles. Here we determine the sulfur isotope effect of the enzyme adenosine phosphosulfate reductase (Apr), which is present in all known organisms conducting MSR and catalyzes the first reductive step in the pathway and reinterpret the sedimentary sulfur isotope record over geological time. Small fractionations may be attributed to low sulfate concentrations and/or high respiration rates, whereas fractionations greater than that of Apr require a low chemical potential at that metabolic step. Since Archean sediments lack fractionation exceeding the Apr value of 20‰, they are indicative of sulfate reducers having had access to ample electron donors to drive their metabolisms. Large fractionations in post-Archean sediments are congruent with a decline of favorable electron donors as aerobic and other high potential metabolic competitors evolved.