The absence of bumblebees on an oceanic island blurs the species boundary of two closely related orchids.

Oceanic islands offer valuable natural laboratories for studying evolution. The Izu Islands, with their recent geological origin, provide an exceptional opportunity to explore the initial evolution on oceanic islands. Another noteworthy aspect is the absence of bumblebee species on most Izu Islands. We used ecological, morphological, and molecular data to investigate the impact of bumblebee absence on the evolution of two closely related orchid species, Goodyera henryi and Goodyera similis, focusing on Kozu Island, the Izu Islands. Our investigation revealed that while G. henryi exclusively relies on a bumblebee species for pollination on the mainland, G. similis is pollinated by scoliid wasps on both the mainland and the island. Intriguingly, all specimens initially categorized as G. henryi on Kozu Island are hybrids of G. henryi and G. similis, leading to the absence of pure G. henryi distribution on the island. These hybrids are pollinated by the scoliid wasp species that also pollinates G. similis on the island. The absence of bumblebees might result in sporadic and inefficient pollination of G. henryi by scoliid wasps, consequently promoting hybrid proliferation on the island. Our findings suggest that the absence of bumblebees can blur plant species boundaries.

Genetic consequences of Last Glacial-Holocene changes in snowfall regime in Arnica mallotopus populations: A plant confined to heavy-snow areas of Japan.

PREMISE: Snow is an important environmental factor affecting plant distribution. Past changes in snowfall regimes may have controlled the demographies of snow-dependent plants. However, our knowledge of changes in the distribution and demographies of such plants is limited because of the lack of fossil records. METHODS: Population genetic and landscape genetic analyses were used to investigate the response of population dynamics of Arnica mallotopus (Asteraceae)-a plant confined to heavy-snow areas of Japan-to changes in snowfall regimes from the Last Glacial Period to the Holocene. RESULTS: The population genetic analysis suggested that the four geographic lineages diverged during the Last Glacial Period. The interaction between reduced snowfall and lower temperatures during this period likely triggered population isolation in separate refugia. Subpopulation differentiation in the northern group was lower than in the southern group. Our ecological niche model predicted that the current distribution was patchy in the southern region; that is, the populations were isolated by topologically flat and climatically unsuitable lowlands. The landscape genetic analysis suggested that areas with little snowfall acted as barriers to the Holocene expansion of species distribution and continued limiting gene flow between local populations. CONCLUSIONS: These findings indicate that postglacial population responses vary among regions and are controlled by environmental and geographic factors. Thus, changes in snowfall regime played a major role in shaping the distribution and genetic structure of the snow-dependent plant.

Second and Outer Coordination Sphere Effects in Nitrogenase, Hydrogenase, Formate Dehydrogenase, and CO Dehydrogenase.

Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.

Host-Shift Speciation Proceeded with Gene Flow in Algae Covering Shells.

AbstractHost shifts represent the advancement of a novel niche and often lead to speciation in symbionts. However, its mechanisms are not well understood. Here, we focused on the alga Pseudocladophora conchopheria growing on the shells of intertidal snails. Previous surveys have shown that the alga has host specificity-only attaching to the shell of Lunella correensis-but we discovered that the alga attaches to the shells of multiple sympatric snails. A genome-wide single-nucleotide polymorphism analysis (MIG-seq) was performed to determine whether host-associated speciation occurred in the algae. As a result, there was no gene flow or limited gene flow among the algae from different hosts, and some algae were genetically differentiated among hosts. In addition, the demographic estimate revealed that speciation with gene flow occurred between the algae from different hosts. Therefore, these results support the idea that host-shift speciation gradually proceeded with gene flow in the algae, providing insight into the early evolution of host shifts.

Phylogeographical evidence for historical long-distance dispersal in the flightless stick insect Ramulus mikado.

Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect Ramulus mikado across Japan, based on a multifaceted molecular approach [cytochrome oxidase subunit I (COI) haplotypes, nuclear simple sequence repeat markers and genome-wide single nucleotide polymorphisms]. Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.

Monotropastrum kirishimense (Ericaceae), a new mycoheterotrophic plant from Japan based on multifaceted evidence.

Due to their reduced morphology, non-photosynthetic plants have been one of the most challenging groups to delimit to species level. The mycoheterotrophic genus Monotropastrum, with the monotypic species M. humile, has been a particularly taxonomically challenging group, owing to its highly reduced vegetative and root morphology. Using integrative species delimitation, we have focused on Japanese Monotropastrum, with a special focus on an unknown taxon with rosy pink petals and sepals. We investigated its flowering phenology, morphology, molecular identity, and associated fungi. Detailed morphological investigation has indicated that it can be distinguished from M. humile by its rosy pink tepals and sepals that are generally more numerous, elliptic, and constantly appressed to the petals throughout its flowering period, and by its obscure root balls that are unified with the surrounding soil, with root tips that hardly protrude. Based on genome-wide single-nucleotide polymorphisms, molecular data has provided clear genetic differentiation between this unknown taxon and M. humile. Monotropastrum humile and this taxon are associated with different Russula lineages, even when they are sympatric. Based on this multifaceted evidence, we describe this unknown taxon as the new species M. kirishimense. Assortative mating resulting from phenological differences has likely contributed to the persistent sympatry between these two species, with distinct mycorrhizal specificity.

Spiranthes hachijoensis (Orchidaceae), a new species within the S. sinensis species complex in Japan, based on morphological, phylogenetic, and ecological evidence.

The systematics of the Old World Spiranthes sinensis (Pers.) Ames species complex (Orchidaceae) has been complicated by its wide distribution and morphological variations. Within the species complex, S. australis Lindl. has been generally accepted as the only Spiranthes Rich. species distributed on the Japanese mainland. The present study provides morphological, phylogenetic, and ecological evidence for the recognition of S. hachijoensis Suetsugu as a new species of the S. sinensis species complex on the Japanese mainland. Spiranthes hachijoensis is morphologically similar to S. hongkongensis S.Y. Hu & Barretto and S. nivea T.P. Lin & W.M. Lin, sharing a degenerated rostellum, pollinia without a viscidium, and distinctly trilobed stigma. However, the taxon can be morphologically distinguished from S. hongkongensis by its glabrous rachis, ovaries, and sepals, and from S. nivea by its papillate labellum disc, larger papillate basal labellum callosities, and glabrous rachis, ovaries, and sepals. The autogamy and flowering phenology (i.e., earlier flowering) of S. hachijoensis are most likely responsible for premating isolation from the sympatric S. australis. A MIG-seq-based high-throughput molecular analysis indicated that the genetic difference between S. hachijoensis and its putative sister species S. sinensis is comparable to, or even greater than, the genetic difference between pairs of other species within the S. sinensis species complex. Our multifaceted approach strongly supports the recognition of S. hachijoensis as a morphologically, phenologically, phylogenetically, and ecologically distinct species.

Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman, crystallography, and DFT calculation.

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or ß subunit contains a ferric heme in the α2ß2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.

Proton Transfer Mechanisms in Bimetallic Hydrogenases.

Hydrogenases are metalloenzymes that catalyze proton reduction and H2 oxidation with outstanding efficiency. They are model systems for bioinorganic chemistry, including low-valent transition metals, hydride chemistry, and proton-coupled electron transfer. In this Account, we describe how photochemistry and infrared difference spectroscopy can be used to identify the dynamic hydrogen-bonding changes that facilitate proton transfer in [NiFe]- and [FeFe]-hydrogenase.[NiFe]-hydrogenase binds a heterobimetallic nickel/iron site embedded in the protein by four cysteine ligands. [FeFe]-hydrogenase carries a homobimetallic iron/iron site attached to the protein by only a single cysteine. Carbon monoxide and cyanide ligands in the active site facilitate detailed investigations of hydrogenase catalysis by infrared spectroscopy because of their strong signals and redox-dependent frequency shifts. We found that specific redox-state transitions in [NiFe]- and [FeFe]-hydrogenase can be triggered by visible light to record extremely sensitive "light-minus-dark" infrared difference spectra monitoring key amino acid residues. As these transitions are coupled to protonation changes, our data allowed investigation of dynamic hydrogen-bonding changes that go well beyond the resolution of protein crystallography.In [NiFe]-hydrogenase, photolysis of the bridging hydride ligand in the Ni-C state was followed by infrared difference spectroscopy. Our data clearly indicate the formation of a protonated cysteine residue as well as hydrogen-bonding changes involving a glutamic acid residue and a "dangling water" molecule. These findings are in excellent agreement with crystallographic analyses of [NiFe]-hydrogenase. In [FeFe]-hydrogenase, an external redox dye was used to accumulate the Hred state. Infrared difference spectra indicate hydrogen-bonding changes involving two glutamic acid residues and a conserved arginine residue. While crystallographic analyses of [FeFe]-hydrogenase in the oxidized state failed to explain the rapid proton transfer because of a breach in the succession of residues, our findings facilitated a precise molecular model of discontinued proton transfer.Comparing both systems, our data emphasize the role of the outer coordination sphere in bimetallic hydrogenases: we suggest that protonation of a nickel-ligating cysteine in [NiFe]-hydrogenase causes the notable preference toward H2 oxidation. On the contrary, proton transfer in [FeFe]-hydrogenase involves an adjacent cysteine as a relay group, promoting both H2 oxidation and proton reduction. These observations may guide the design of organometallic compounds that mimic the catalytic properties of hydrogenases.

Evolutionary history of Hemerocallis in Japan inferred from chloroplast and nuclear phylogenies and levels of interspecific gene flow.

The perennial herb genus Hemerocallis (Asphodelaceae) shows four flowering types: diurnal half-day, diurnal one-day, nocturnal half-day, and nocturnal one-day flowering. These flowering types are corresponding to their main pollinators, and probably act as a primary mechanism of reproductive isolation. To examine how the four flowering types diverged, we reconstructed the phylogeny of the Japanese species of Hemerocallis using 1615 loci of nuclear genome-wide SNPs and 2078 bp sequences of four cpDNA regions. We also examined interspecific gene flows among taxa by an Isolation-with-Migration model and a population structure analysis. Our study revealed an inconsistency between chloroplast and nuclear genome phylogenies, which may have resulted from chloroplast capture. Each of the following five clusters is monophyletic and clearly separated on the nuclear genome-wide phylogenetic tree: (I) two nocturnal flowering species with lemon-yellow flowers, H. citrina (half-day flowering) and H. lilioasphodelus (one-day flowering); (II) a diurnal one-day flowering species with yellow-orange flowers, H. middendorffii; (III) a variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. disticha; (IV) another variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. aurantiaca, and a diurnal one-day flowering species with yellow-orange flowers, H. major; (V) a diurnal half-day flowering species with yellow-orange flowers, H. hakuunensis. The five clusters are consistent with traditional phenotype-based taxonomy (cluster I, cluster II, and clusters III-V correspond to Hemerocallis sect. Hemerocallis, Capitatae, and Fulvae, respectively). These findings could indicate that three flowering types (nocturnal flowering, diurnal one-day flowering, and diurnal half-day flowering) diverged in early evolutionary stages of Hemerocallis and subsequently a change from diurnal half-day flowering to diurnal one-day flowering occurred in a lineage of H. major. While genetic differentiation among the five clusters was well maintained, significant gene flow was detected between most pairs of taxa, suggesting that repeated hybridization played a role in the evolution of those taxa.

A strategic sampling design revealed the local genetic structure of cold-water fluvial sculpin: a focus on groundwater-dependent water temperature heterogeneity.

A key piece of information for ecosystem management is the relationship between the environment and population genetic structure. However, it is difficult to clearly quantify the effects of environmental factors on genetic differentiation because of spatial autocorrelation and analytical problems. In this study, we focused on stream ecosystems and the environmental heterogeneity caused by groundwater and constructed a sampling design in which geographic distance and environmental differences are not correlated. Using multiplexed ISSR genotyping by sequencing (MIG-seq) method, a fine-scale population genetics study was conducted in fluvial sculpin Cottus nozawae, for which summer water temperature is the determinant factor in distribution and survival. There was a clear genetic structure in the watershed. Although a significant isolation-by-distance pattern was detected in the watershed, there was no association between genetic differentiation and water temperature. Instead, asymmetric gene flow from relatively low-temperature streams to high-temperature streams was detected, indicating the importance of low-temperature streams and continuous habitats. The groundwater-focused sampling strategy yielded insightful results for conservation.

Comparative analysis of spatial genetic structures in sympatric populations of two riparian plants, Saxifraga acerifolia and Saxifraga fortunei.

PREMISE: The genetic structure between plant populations is facilitated by the spatial population arrangement and limited dispersal of seed and pollen. Saxifraga acerifolia, a local endemic species in Japan, is a habitat specialist that is confined to waterfalls in riparian environments. Its sister species, Saxifraga fortunei, is a generalist that is widely distributed along riverbanks. Here, we examined sympatric populations of the two Saxifraga species to test whether the differences in habitat preference and colonization process influenced regional and local genetic structures. METHODS: To reveal genetic structures, we examined chloroplast microsatellite variations and genome-wide nucleotide polymorphisms obtained by genotyping by sequencing. We also estimated the gene flow among and within populations and performed landscape genetic analyses to evaluate seed and pollen movement and the extent of genetic isolation related to geographic distance and/or habitat differences. RESULTS: We found strong genetic structure in the specialist S. acerifolia, even on a small spatial scale (<1 km part); each population on a different waterfall in one river system had a completely different predominant haplotype. By contrast, the generalist S. fortunei showed no clear genetic differentiation. CONCLUSIONS: Our findings suggest that the level of genetic isolation was increased in S. acerifolia by the spatially fragmented habitat and limited seed and pollen dispersal over waterfalls. Habitat differentiation between the sister taxa could have contributed to the different patterns of gene flow and then shaped the contrasting genetic structures.

Mechanism and Application of the Catalytic Reaction of [NiFe] Hydrogenase: Recent Developments.

Hydrogenases (H2 ase) catalyze the oxidation of dihydrogen and the reduction of protons with remarkable efficiency, thereby attracting considerable attention in the energy field due to their biotechnological potential. For this simple reaction, [NiFe] H2 ase has developed a sophisticated but intricate mechanism with the heterolytic cleavage of dihydrogen, where its Ni-Fe active site exhibits various redox states. Recently, new spectroscopic and crystal structure studies of [NiFe] H2 ases have been reported, providing significant insights into the catalytic reaction mechanism, hydrophobic gas-access tunnel, proton-transfer pathway, and electron-transfer pathway of [NiFe] H2 ases. In addition, [NiFe] H2 ases have been shown to play an important role in biofuel cell and solar dihydrogen production. This concept provides an overview of the biocatalytic reaction mechanism and biochemical application of [NiFe] H2 ases based on the new findings.

Regioselective Chemical Modification of Cysteine Residues on Protein Surfaces Focusing on Local Environment around the Conjugation Site.

For chemical modification of cysteines in a protein, the regioselectivity among cysteine residues on the protein surface is an issue to be considered. To elucidate the determinants of cysteine reactivities on protein surfaces, we have investigated the chemical modification of the adenylate kinase A55C/C77S/V169C mutant as an experimental model. Although Cys55 and Cys169 are commonly located on the protein surface, Cys55 showed the ca. 3-6-fold higher reactivity compared to Cys169 in a reaction with a pyrene derivative. By a further conjugation of a phenanthroline derivative into the vacant Cys thiol, fluorescence quenching was attained by a pyrene-phenanthroline interaction that occurred by the conformational change of the protein. The K50A mutation further enhanced the regioselectivity of pyrene conjugation in Cys55, which is attributed to the effects of structural flexibility in the vicinity of Cys55 on its reactivity. To regioselectively conjugate different types of synthetic molecules onto the surface of a protein, perturbation in the local structural flexibility around the conjugation sites will be a useful strategy.

Domain-Swapping Design by Polyproline Rod Insertion.

During domain swapping, proteins mutually interconvert structural elements to form a di-/oligomer. Engineering this process by design is important for creating a higher order protein assembly with minimal modification. Herein, a simple design strategy is shown for domain-swapping formation by loop deletion and insertion of a polyproline rod. Crystal structures revealed the formation of the domain-swapped dimers and polyproline portion formed a polyproline II (PPII) structure. Small-angle X-ray scattering demonstrated that an extended orientation of domain-swapped dimer was retained in solution. It is found that a multiple of three of inserting proline residue is favored for domain swapping because of the helical nature of PPII. The rigid nature of the polyproline rod enables precise control of the interdomain distance and orientation.

Cysteine SH and Glutamate COOH Contributions to [NiFe] Hydrogenase Proton Transfer Revealed by Highly Sensitive FTIR Spectroscopy.

A [NiFe] hydrogenase (H2 ase) is a proton-coupled electron transfer enzyme that catalyses reversible H2 oxidation; however, its fundamental proton transfer pathway remains unknown. Herein, we observed the protonation of Cys546-SH and Glu34-COOH near the Ni-Fe site with high-sensitivity infrared difference spectra by utilizing Ni-C-to-Ni-L and Ni-C-to-Ni-SIa photoconversions. Protonated Cys546-SH in the Ni-L state was verified by the observed SH stretching frequency (2505 cm-1 ), whereas Cys546 was deprotonated in the Ni-C and Ni-SIa states. Glu34-COOH was double H-bonded in the Ni-L state, as determined by the COOH stretching frequency (1700 cm-1 ), and single H-bonded in the Ni-C and Ni-SIa states. Additionally, a stretching mode of an ordered water molecule was observed in the Ni-L and Ni-C states. These results elucidate the organized proton transfer pathway during the catalytic reaction of a [NiFe] H2 ase, which is regulated by the H-bond network of Cys546, Glu34, and an ordered water molecule.

Design of artificial metalloproteins/metalloenzymes by tuning noncovalent interactions.

Noncovalent weak interactions [hydrophobic interaction and hydrogen (H)-bond] play crucial roles in controlling the functions of biomolecules, and thus have been used to design artificial metalloproteins/metalloenzymes during the past few decades. In this review, we focus on the recent progresses in protein design by tuning the noncovalent interactions, including hydrophobic and H-bonding interactions. The topics include redesign and reuse of the heme pocket and other protein scaffolds, design of the heme protein interface, and de novo design of metalloproteins. The informations not only give insights into the metalloenzyme reaction mechanisms but also provide new reactions for future applications.

Global Structural Flexibility of Metalloproteins Regulates Reactivity of Transition Metal Ion in the Protein Core: An Experimental Study Using Thiol-subtilisin as a Model Protein.

The functions of metal-containing proteins (metalloproteins) are determined by the reactivities of transition metal ions at their active sites. Because protein macromolecular structures have several molecular degrees of freedom, global structural flexibility may also regulate the properties of metalloproteins. However, the influence of this factor has not been fully delineated in mechanistic studies of metalloproteins. Accordingly, we have investigated the relationship between global protein flexibility and the characteristics of a transition metal ion in the protein core using thiol-subtilisin (tSTL) with a Cys-coordinated Cu2+ ion as a model system. Although tSTL has two Ca2+ -binding sites, the Ca2+ -binding status hardly affects its secondary structure. Nevertheless, guanidinium-induced denaturation and amide H/D exchange indicated the increase in the structural flexibility of tSTL by the removal of bound Ca2+ ions. Electron paramagnetic resonance and absorption spectral changes have revealed that the protein flexibility determines the characteristics of a Cu2+ ion in tSTL. Therefore, global protein flexibility should be recognized as an important factor that regulates the properties of metalloproteins.

Synergistic Effect of Distal Polar Interactions in Myoglobin and Their Structural Consequences.

In the L29F variant of myoglobin (Mb), the coordination of oxygen (O2) to the heme Fe atom is stabilized by favorable electrostatic interactions between the polar Fe-O2 moiety and the multipole of the phenyl ring of the Phe29 side chain (Phe29 interaction), in addition to the well-known hydrogen bond (H-bond) between the Fe-bound O2 and the 64th residue (distal H-bond; Carver, T. E.; Brantley, R. E., Jr.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem. 1992, 267, 14443-14450). The O2 and carbon monoxide (CO) binding properties and autoxidation of the L29F/H64L and L29F/H64Q variants reconstituted with a series of chemically modified heme cofactors were analyzed and then compared with those of native Mb, and the L29F, H64Q, and H64L variants similarly reconstituted with the chemically modified heme cofactors in order to elucidate the relationship between the Phe29 interaction and the distal H-bond that critically contributes to stabilization of Fe-bound O2. We found that the Phe29 interaction and distal H-bond act cooperatively to stabilize the Fe-bound O2 in such a manner that the Phe29 interaction strengthens with increasing strength of the distal H-bond. Comparison of the functional properties between the L29F and H64L variants indicated that the synergistic effect of the two interactions decreases the O2 dissociation and autoxidation rate constants of the protein by factors of ∼1/2000 and ∼1/400, respectively. Although the CO binding properties of the proteins were not greatly affected by the distal polar interactions, their synergistic effects were clearly and sharply manifested in the vibrational frequencies of the Fe-bound C-O stretching of the proteins.

Theoretical analysis of the domain-swapped dimerization of cytochrome c: An MD and 3D-RISM approach.

The structural stability of a cytochrome c domain-swapped dimer compared with that of the monomer was investigated by molecular dynamics (MD) simulations and by three-dimensional reference interaction site model (3D-RISM) theory. The structural fluctuation and structural energy of cytochrome c were treated by MD simulations, and the solvation thermodynamics was treated by 3D-RISM theory. The domain-swapped dimer state is slightly less stable than the monomer state, which is consistent with experimental observations; the total free energy difference is calculated as 25 kcal mol-1. The conformational change and translational/rotational entropy change contribute to the destabilization of the dimer, whereas the hydration and vibrational entropy contribute to the stabilization. Further analyses on the residues located at the hinge loop for swapping were conducted, and the results reveal details at the molecular level of the structural and interaction changes upon dimerization.