FT-IR Characterization of the Light-Induced Ni-L2 and Ni-L3 States of [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F

Different light-induced Ni-L states of [NiFe] hydrogenase from its Ni-C state have previously been observed by EPR spectroscopy. Herein, we succeeded in detecting simultaneously two Ni-L states of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F by FT-IR spectroscopy. A new light-induced ν_CO band at 1890 cm^–1 and ν_CN bands at 2034 and 2047 cm^–1 were detected in the FT-IR spectra of the H₂-activated enzyme under N₂ atmosphere at basic conditions, in addition to the 1910 cm^–1 ν_CO band and 2047 and 2061 cm^–1 ν_CN bands of the Ni-L2 state. The new bands were attributed to the Ni-L3 state by comparison of the FT-IR and EPR spectra. The ν_CO and ν_CN frequencies of the Ni-L3 state are the lowest frequencies observed among the corresponding frequencies of standard-type [NiFe] hydrogenases in various redox states. These results indicate that a residue, presumably Ni-coordinating Cys546, is protonated and deprotonated in the Ni-L2 and Ni-L3 states, respectively. Relatively small Δ<i>H</i> (6.4 ± 0.8 kJ mol^–1) and Δ<i>S</i> (25.5 ± 10.3 J mol^–1 K^–1) values were obtained for the conversion from the Ni-L2 to Ni-L3 state, which was in agreement with the previous proposals that deprotonation of Cys546 is important for the catalytic reaction of the enzyme

Domain Swapping of the Heme and N‑Terminal α‑Helix in <i>Hydrogenobacter thermophilus</i> Cytochrome <i>c</i>₅₅₂ Dimer

Oxidized horse cytochrome <i>c</i> (cyt <i>c</i>) has been shown to oligomerize by domain swapping its C-terminal helix successively. We show that the structural and thermodynamic properties of dimeric <i>Hydrogenobacter thermophilus</i> (HT) cytochrome <i>c</i>₅₅₂ (cyt <i>c</i>₅₅₂) and dimeric horse cyt <i>c</i> are different, although both proteins belong to the cyt <i>c</i> superfamily. Optical absorption and circular dichroism spectra of oxidized dimeric HT cyt <i>c</i>₅₅₂ were identical to the corresponding spectra of its monomer. Dimeric HT cyt <i>c</i>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 <i>c</i>₅₅₂. The packing of the amino acid residues important for thermostability in monomeric HT cyt <i>c</i>₅₅₂ were maintained in its dimer, and thus, dimeric HT cyt <i>c</i>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

CD spectra and small angle X-ray scattering curves of WT and M61A PA cyt <i>c</i>₅₅₁.

<p>(A) CD spectra of oxidized monomeric WT (red) and M61A (green) PA cyt <i>c</i>₅₅₁. Measurement conditions: Sample concentration, 10 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, room temperature. (B) Small angle X-ray scattering curves of oxidized monomeric WT (red) and M61A (green) PA cyt <i>c</i>₅₅₁ shown by Kratky plots. The intensities are normalized at their maximum intensities. Measurement conditions: sample concentration, 500 μM (heme unit); buffer, 50 mM potassium phosphate buffer; pH, 7.0; temperature, 20°C.</p

Fe–His16 and Fe–Met61 distances in monomeric and dimeric WT PA cyt <i>c</i>₅₅₁.

<p>^a PDB ID: 351C.</p><p>^b There are two independent WT PA cyt <i>c</i>₅₅₁ molecules in the asymmetric unit of dimeric WT PA cyt <i>c</i>₅₅₁ crystal.</p><p>Fe–His16 and Fe–Met61 distances in monomeric and dimeric WT PA cyt <i>c</i>₅₅₁.</p

Active site structures of monomeric and dimeric WT PA cyt <i>c</i>₅₅₁.

<p>(A) Structure of monomeric WT PA cyt <i>c</i>₅₅₁ (PDB ID: 351C). (B) Structure of dimeric WT PA cyt <i>c</i>₅₅₁ (PDB ID: 3X39). The heme and side-chains of amino acid residues near the heme (Phe7, Cys12, Ala14, Cys15, His16, Val23, Pro25, Val30, Leu44, Arg47, Ile48, Ser52, Trp56, Pro60, Met61, Pro62, Pro63, Asn64, Leu74, and Val78) are shown as stick models. The sulfur atoms of the heme axial Met ligand and heme-linked Cys are shown in yellow, and the nitrogen atoms of the heme axial His ligand are shown in blue. The cyan strand in the dimeric structure is a region from another molecule. The hemes and Thr20–Met22 residues (hinge loop) are depicted in dark and pale colors, respectively.</p

Refined Regio- and Stereoselective Hydroxylation of l‑Pipecolic Acid by Protein Engineering of l‑Proline <i>cis</i>-4-Hydroxylase Based on the X‑ray Crystal Structure

Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe²⁺/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of l-proline into hydroxy-l-prolines (Hyps). l-Proline <i>cis</i>-4-hydroxylases (<i>cis</i>-P4Hs) from <i>Sinorhizobium meliloti</i> and <i>Mesorhizobium loti</i> catalyze the hydroxylation of l-proline, generating <i>cis</i>-4-hydroxy-l-proline, as well as the hydroxylation of l-pipecolic acid (l-Pip), generating two regioisomers, <i>cis</i>-5-Hypip and <i>cis</i>-3-Hypip. To selectively produce <i>cis</i>-5-Hypip without simultaneous production of two isomers, protein engineering of <i>cis</i>-P4Hs is required. We therefore carried out protein engineering of <i>cis</i>-P4H to facilitate the conversion of the majority of l-Pip into the <i>cis</i>-5-Hypip isomer. We first solved the X-ray crystal structure of <i>cis</i>-P4H in complex with each of l-Pro and l-Pip. Then, we conducted three rounds of directed evolution and successfully created a <i>cis</i>-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward <i>cis</i>-5-Hypip

Formation of Oligomeric Cytochrome <i>c</i> during Folding by Intermolecular Hydrophobic Interaction between N- and C‑Terminal α‑Helices

We have previously shown that horse cytochrome <i>c</i> (cyt <i>c</i>) forms oligomers by domain swapping its C-terminal α-helix when interacting with ethanol. Although folding of cyt <i>c</i> has been studied extensively, formation of domain-swapped oligomers of cyt <i>c</i> during folding has never been reported. We found that domain-swapped oligomeric cyt <i>c</i> is produced during refolding from its guanidinium ion-induced unfolded state at high protein concentrations and low temperatures. The obtained dimer exhibited a domain-swapped structure exchanging the C-terminal α-helical region between molecules. The extent of dimer formation decreased significantly for the folding of C-terminal cyt <i>c</i> mutants with reduced hydrophobicity achieved by replacement of hydrophobic residues with Gly in the C-terminal region, whereas a large amount of heterodimers was generated for the folding of a mixture of N- and C-terminal mutants. These results show that cyt <i>c</i> oligomers are formed through intermolecular hydrophobic interaction between the N- and C-terminal α-helices during folding. A slow phase (4–5 s) was observed in addition to a 400–500 ms phase during folding of a high concentration of cyt <i>c</i> in the presence of 1.17 M guanidine hydrochloride. The fast phase is attributed to the intramolecular ligand exchange process, and we attribute the slow phase to the ligand exchange process in oligomers. These results show that it is important to consider formation of domain-swapped oligomeric proteins when folding at high protein concentrations

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 <i>Desulfovibrio vulgaris</i> 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

Formation of Domain-Swapped Oligomer of Cytochrome <i>c</i> from Its Molten Globule State Oligomer

Many proteins, including cytochrome <i>c</i> (cyt <i>c</i>), have been shown to form domain-swapped oligomers, but the factors governing the oligomerization process remain unrevealed. We obtained oligomers of cyt <i>c</i> by refolding cyt <i>c</i> from its acid molten globule state to neutral pH state under high protein and ion concentrations. The amount of oligomeric cyt <i>c</i> obtained depended on the nature of the anion (chaotropic or kosmotropic) in the solution: ClO₄^– (oligomers, 11% ± 2% (heme unit)), SCN^– (10% ± 2%), I^– (6% ± 2%), NO₃^– (3% ± 1%), Br^– (2% ± 1%), Cl^– (2% ± 1%), and SO₄^2– (3% ± 1%) for refolding of 2 mM cyt <i>c</i> (anion concentration 125 mM). Dimeric cyt <i>c</i> obtained by refolding from the molten globule state exhibited a domain-swapped structure, in which the C-terminal α-helices were exchanged between protomers. According to small-angle X-ray scattering measurements, approximately 25% of the cyt <i>c</i> molecules were dimerized in the molten globule state containing 125 mM ClO₄^–. These results indicate that a certain amount of molten globule state oligomers of cyt <i>c</i> convert to domain-swapped oligomers during refolding and that the intermolecular interactions necessary for domain swapping are present in the molten globule state