Domain-swapped dimer of Pseudomonas aeruginosa cytochrome c551: structural insights into domain swapping of cytochrome c family proteins.

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c551 (PA cyt c551), and Hydrogenobacter thermophilus cytochrome c552 (HT cyt c552), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met-heme coordination significantly compared to the monomer. HT cyt c552 forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c551 also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c551 were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.

Crystallization and preliminary X-ray analysis of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1.

NAD+-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD+ and NADH. Here, the isolation, purification and crystallization of the NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD+-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58 Šresolution and belonged to space group C2, with unit-cell parameters a=131.43, b=189.71, c=124.59 Å, ß=109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, VM was calculated to be 2.2 Å3 Da(-1), which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.

Light-Driven Hydrogen Production by Hydrogenases and a Ru-Complex inside a Nanoporous Glass Plate under Aerobic External Conditions.

Hydrogenases are powerful catalysts for light-driven H2 production using a combination of photosensitizers. However, except oxygen-tolerant hydrogenases, they are immediately deactivated under aerobic conditions. We report a light-driven H2 evolution system that works stably even under aerobic conditions. A [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F was immobilized inside nanoporous glass plates (PGPs) with a pore diameter of 50 nm together with a ruthenium complex and methyl viologen as a photosensitizer and an electron mediator, respectively. After immersion of PGP into the medium containing the catalytic components, an anaerobic environment automatically established inside the nanopores even under aerobic external conditions upon irradiation with solar-simulated light; this system constantly evolved H2 with an efficiency of 3.7 µmol H2 m(-2) s(-1). The PGP system proposed in this work represents a promising first step toward the development of an O2-tolerant solar energy conversion system.

Photosensitivity of the Ni-A state of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F with visible light.

[NiFe] hydrogenase catalyzes reversible oxidation of molecular hydrogen. Its active site is constructed of a hetero dinuclear Ni-Fe complex, and the oxidation state of the Ni ion changes according to the redox state of the enzyme. We found that the Ni-A state (an inactive unready, oxidized state) of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (DvMF) is light sensitive and forms a new state (Ni-AL) with irradiation of visible light. The Fourier transform infrared (FT-IR) bands at 1956, 2084 and 2094 cm(-1) of the Ni-A state shifted to 1971, 2086 and 2098 cm(-1) in the Ni-AL state. The g-values of g(x)=2.30, g(y)=2.23 and g(z)=2.01 for the signals in the electron paramagnetic resonance (EPR) spectrum of the Ni-A state at room temperature varied for -0.009, +0.012 and +0.010, respectively, upon light irradiation. The light-induced Ni-AL state converted back immediately to the Ni-A state under dark condition at room temperature. These results show that the coordination structure of the Fe site of the Ni-A state of [NiFe] hydrogenase is perturbed significantly by light irradiation with relatively small coordination change at the Ni site.

Domain swapping of the heme and N-terminal α-helix in Hydrogenobacter thermophilus cytochrome c(552) dimer.

Oxidized horse cytochrome c (cyt c) has been shown to oligomerize by domain swapping its C-terminal helix successively. We show that the structural and thermodynamic properties of dimeric Hydrogenobacter thermophilus (HT) cytochrome c(552) (cyt c(552)) and dimeric horse cyt c are different, although both proteins belong to the cyt c superfamily. Optical absorption and circular dichroism spectra of oxidized dimeric HT cyt c(552) were identical to the corresponding spectra of its monomer. Dimeric HT cyt c(552) exhibited a domain-swapped structure, where the N-terminal α-helix together with the heme was exchanged between protomers. Since a relatively strong H-bond network was formed at the loop around the heme-coordinating Met, the C-terminal α-helix did not dissociate from the rest of the protein in dimeric HT cyt c(552). The packing of the amino acid residues important for thermostability in monomeric HT cyt c(552) were maintained in its dimer, and thus, dimeric HT cyt c(552) exhibited high thermostability. Although the midpoint redox potential shifted from 240 ± 2 to 213 ± 2 mV by dimerization, it was maintained relatively high. Ethanol has been shown to decrease both the activation enthalpy and activation entropy for the dissociation of the dimer to monomers from 140 ± 9 to 110 ± 5 kcal/mol and 310 ± 30 to 270 ± 20 cal/(mol·K), respectively. Enthalpy change for the dissociation of the dimer to monomers was positive (14 ± 2 kcal/mol per protomer unit). These results give new insights into factors governing the swapping region and thermodynamic properties of domain swapping.

Structural and oxygen binding properties of dimeric horse myoglobin.

Myoglobin (Mb) stores dioxygen in muscles, and is a fundamental model protein widely used in molecular design. The presence of dimeric Mb has been known for more than forty years, but its structural and oxygen binding properties remain unknown. From an X-ray crystallographic analysis at 1.05 Å resolution, we found that dimeric metMb exhibits a domain-swapped structure with two extended α-helices. Each new long α-helix is formed by the E and F helices and the EF-loop of the original monomer, and as a result the proximal and distal histidines of the heme originate from different protomers. The heme orientation in the dimer was in the normal mode as in the monomer, but regulated faster from the reverse to normal orientation. The dimer possessed the oxygen binding property, although it exhibited a slightly higher oxygen binding affinity (∼1.4 fold) compared to the monomer and showed no cooperativity for oxygen binding. The oxygen binding rate constant (k(on)) of the dimer ((14.0 ± 0.7) × 10(6) M(-1) s(-1)) was similar to that of the monomer, whereas the oxygen dissociation rate constant (k(off)) of the dimer (8 ± 1 s(-1)) was smaller than that of the monomer (12 ± 1 s(-1)). We attribute the similar k(on) values to their active site structures being similar, whereas the faster regulation of the heme orientation and the smaller k(off) in the dimer are presumably due to the slight change in the active site structure and/or more rigid structure compared to the monomer. These results show that domain swapping may be a new tool for protein engineering.