Structure and function relationship of formate dehydrogenases: an overview of recent progress.

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.

Structural and spectroscopic characterization of CO inhibition of [NiFe]-hydrogenase from Citrobacter sp. S-77.

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 Citrobacter 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 Šresolution 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.

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.

Crystallization and preliminary X-ray analysis of dimeric and trimeric cytochromes c from horse heart.

Cytochrome c (cyt c) is an electron-transfer protein in the respiratory chain of mitochondria. It is known to form polymers, but its polymerization mechanism is still unknown. Dimeric and trimeric cyt c from horse were successfully crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol as a precipitating reagent. The crystal of dimeric cyt c belonged to space group P1, with unit-cell parameters a = 41.8, b = 56.3, c = 60.8 Å, α = 66.3, ß = 89.9, γ = 73.7°, whereas that of trimeric cyt c belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.2, b = 95.7, c = 130.9 Å. Initial structure models showed that the crystals of dimeric and trimeric cyt c contained two dimers and two trimers, respectively, in the asymmetric unit.

Cytochrome c polymerization by successive domain swapping at the C-terminal helix.

Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and (NH(4))(2)HPO(4). The absorption and CD spectra of high-order oligomers (approximately 40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the DeltaH of the oligomer dissociation to monomers was estimated to be about -20 kcal/mol per protomer unit, where Met-heme coordination appears to contribute largely to DeltaH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.

High-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center.

A high-throughput crystallization-to-structure pipeline for structural genomics was recently developed at the Advanced Protein Crystallography Research Group of the RIKEN SPring-8 Center in Japan. The structure determination pipeline includes three newly developed technologies for automating X-ray protein crystallography: the automated crystallization and observation robot system "TERA", the SPring-8 Precise Automatic Cryosample Exchanger "SPACE" for automated data collection, and the Package of Expert Researcher's Operation Network "PERON" for automated crystallographic computation from phasing to model checking. During the 5 years following April, 2002, this pipeline was used by seven researchers to determine 138 independent crystal structures (resulting from 437 purified proteins, 234 cryoloop-mountable crystals, and 175 diffraction data sets). The protocols used in the high-throughput pipeline are described in this paper.