Complex structure of NADPH-cytochrome P450 reductase and heme oxygenase-1

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to FAD in CPR, then to FMN in CPR, and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, ``closed-open transition'' of CPR is indispensable because FMN in the closed conformation of CPR is covered by FAD-binding domain, thus FMN is not exposed to the surface in the closed conformation. Recently, Hamdane et al. determined the crystal structures of a hinge-shortened rat CPR variant (ΔTGEE), which favors an open conformation [1]. In the open conformation of CPR, FMN is exposed to the surface, thus this conformation appears to be favorable to interact with the redox partners, though no complex structure of CPR and its redox partner has been determined. Here, we demonstrate that ΔTGEE makes a stable complex with heme-rat HO-1 (rHO-1) complex and can support HO reaction, though its efficiency is extremely limited. Further we determine the crystal structure of ΔTGEE in complex with heme-rHO-1 at 4.3 Å resolution [2]. X-ray scattering and biochemical data suggest that the complex structure of ΔTGEE and heme-rHO-1 is similar to that of wild type CPR and heme-rHO-1. Distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme. On the other hand, FAD is far from FMN and heme, indicating that the ``closed-open transition'' of CPR is required for electron transfer from FAD to FMN.

Crystal structures of γ-glutamyltranspeptidase in complex with inhibitors

γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) is involved in the degradation of γ-glutamyl compounds such as glutathione (GSH; γ-glutamyl-cysteinyl-glycine) . A major physiological role of this enzyme is to cleave the extracellular GSH as a source of cysteine for intracellular glutathione biosynthesis. Another crucial role of GGT is to cleave glutathione-S-conjugates as a key step in detoxification of xenobiotics and drug metabolism. In mammals, GGT has been implicated in physiological disorders such as Parkinson's disease, other neurodegenerative diseases including Alzheimer's disease and cardiovascular disease. The indispensable roles played by GGT in GSH-mediated detoxification and cellular response to oxidative stress suggest that GGT is an attractive pharmaceutical target. We here report the binding mode of acivicin, a well-known glutamine antagonist, to B. subtilis GGT at 1.8 Å resolution showing that acivicin is bound to the Oγ atom of Thr403, the catalytic nucleophile of the enzyme, through its C3 atom [1]. The observed electron density around the C3 atom was best fitted to the planar and sp2 hybridized carbon, consistent with a simple nucleophilic substitution of Cl at the imino carbon by Oγ atom of Thr403. Furthermore, comparison of three bacterial enzymes, the GGTs from E. coli, H. pylori and B. subtilis in complex with acivicin, showed significant diversity in the orientation of the dihydroisoxazole ring among three GGTs. The differences are discussed in terms of the recognition of the α-amino and α-carboxy groups in preference to the dihydroisoxazole ring as observed in time-lapse soaking crystal structures of B. subtilis GGT with acivicin.

Neutron Structure of Phycocyanobilin:oxidoreductase Complexed with Biliverdin

Phytobilins are linear tetrapyrrole compounds used as chromophore for light harvesting and photoreceptor proteins in higher plants, algae, and cyanobacteria. Phytobilins are synthesized from biliverdin IX(alpha) (BV). Phycocianobilin:oxidoreductase (PcyA) is an enzyme to produce phycocyanobilin (PCB) used as chromophore for light harvesting and photoreceptor proteins. PcyA is unique because it catalyzes the reduction of BV by two sequential steps; the first step is the reduction of the vinyl of the BV D-ring to produce 18(1)-18(2)-dihydrobiliverdin (18EtBV), and the second step is the reduction of the A-ring. In these reduction steps, four hydrogen atoms are delivered to BV. The earlier studies showed that the carboxyl group of Asp105 showed dual conformations. This has been attributed to the difference of its protonation states. The catalytically essential His88 was suggested to be protonated (i. e. His88 is a proton donor) to donate the proton to BV. BVH+ (N-protonated) state, in which four pyrrole N atoms of BV were fully protonated, was proposed to be partially formed when BV was bound to PcyA. Further, another tautomeric BVH+ state in which three of four pyrrole N atoms of BV were protonated and the lactam (C=O) group of BV D-ring was protonated as lactim (C-OH; O-protonated) was proposed. Additionally, newly identified water molecule near BV has been suggested to be a proton donor. To elucidate the H atom positions of these molecules, we determined the neutron crystal structure of the PcyA-BV complex at 1.95 Å resolution. Crystal with approximately 2.2 X 1.8 X 0.8 mm3 size, which was soaked into the deuterium-exchanged crystallization solution, was used in the diffraction experiment. The neutron diffraction intensity data was collected using IBARAKI Biological Crystal Diffractometer (iBIX) in J-PARC. In this conference, we report the protonation states of catalytically important residues and BV as well as orientations of water molecules in the PcyA-BV complex.

Structure determination of two-dimensional atomic sheet of silicene using TRHEPD

This study reports determination of the atomic coordinates of a two-dimensional atomic sheet, silicene, by using total reflection high-energy positron diffraction (TRHEPD) [1]. TRHEPD method, formerly called as RHEPD, is a surface-sensitive tool owing to the total reflection of positrons. Since the sign of the potential energy for positrons in crystals is positive, opposite to that for the electrons, the positron beam at a grazing incidence are totally reflected at a crystal surface. The penetration depth of the positron beam in the total reflection region is estimated to be approximately a few Å, which corresponds to the thickness of 1-2 atomic layers. Thus, the positron beam selectively sees the topmost surface layer and hence the TRHEPD method is very useful for structure determinations of crystal surface and two-dimensional atomic sheet on the substrate. Silicene is a two-dimensional atomic sheet of silicon. Since the silicene has an intriguing property such as a Dirac cone like a graphene, it attracts increasing attention as a candidate for future devices. Recently, the synthesis of silicene on a Ag(111) surface was successfully performed [2]. Although the atomic coordinates of the silicene in this system was theoretically calculated, they were not confirmed experimentally. It is very important to experimentally determine the magnitude of the buckling in silicene and the spacing between the bottom of the silicene and the substrate because the dispersion of the Dirac cone is closely related to these structure parameters. We thus investigated the atomic positions of the silicene on the Ag(111) surface using the TRHEPD [3]. From the rocking curve analysis based on the dynamical diffraction theory of positrons (see figure), the existence of the buckling (0.83 Å) in silicene was verified experimentally. Moreover, the spacing between the silicene and the substrate was determined as 2.17 Å. The structural difference with the graphene will be also discussed.

Surface sensitivity of total reflection high-energy positron diffraction

"Reflection high-energy positron diffraction (RHEPD) is the positron counterpart of reflection high-energy electron diffraction (RHEED). RHEPD was proposed in 1992 [1], and first demonstrated in 1998 [2]. Unlike the case of the electron, the potential energy of the positron inside a crystal is positive, and hence positrons incident on a crystal surface with a glancing angle smaller than a certain critical angle are totally reflected. This feature makes the positrons a tool extremely sensitive to the topmost layer of the crystal surface. Recent development of a brightness-enhanced intense positron beam at KEK [K. Wada, et al., J. Phys.: Conf. Ser. 443, 012082 (2013)] has made it possible to obtain clear RHEPD patterns. We rename the technique with a refined beam as ""total reflection high-energy positron diffraction (TRHEPD)"". Here we demonstrate that the TRHEPD pattern from the Si(111)-7x7 DAS surface taken with a glancing angle smaller than the critical angle for the total reflection is essentially determined only by the atoms exposed on the surface (adatoms and the atoms in the first surface layer) [3]. The technical details of the positron beam preparation [M. Maekawa, et al., to be published in Eur. Phys. J. D (2014)], results on the Pt/Ge(001) nano-wire surface[I. Mochizuki, et al., Phys. Rev. B 85, 245438 (2012)], TiO2(110)-1x2 surface, and silicene on Ag(111) surface [Y. Fukaya, et al., Phys. Rev. B 88 205413 (2013)] are also presented in this conference. "

TRHEPD rocking curve analyses of Pt/Ge(001) and TiO₂(110) surfaces

Recently, we developed new total reflection high-energy positron diffraction (TRHEPD) apparatus [1] on a beam line of the linac-based intense positron beam of the Slow Positron Facility at KEK, Japan. The high intensity allows us to install a brightness-enhancement section, which to observation of clear positron diffraction patterns for crystal surfaces under total reflection condition. In this work, we investigated the atomic configuration of Pt-induced nanowires formed on a Ge(001) surface [2] using the apparatus. By means of the diffraction intensity analysis based on the dynamical diffraction theory, or TRHEPD rocking curve analysis, a previously proposed theoretical model [D. E. P. Vanpoucke et al., Phys. Rev. B 77, 241308(R) (2008)], composed of Ge dimers on the top layer and buried Pt arrays in the second and fourth layers, was confirmed to be the fundamental structure of the nanowire. We also investigated the atomic configuration of a rutile-TiO₂ (110) surface. It is well known that the structure of this surface transforms its periodicity from (1×1) to (1×2) by elevating the sample temperature above ~1100 K, whereas the detailed structure is yet to be revealed. There is a longstanding controversy between the structure models proposed by scanning tunneling microscopy, low energy electron diffraction, surface X-ray diffraction, first-principles calculation with density functional theory results, etc. To solve the problem, we have measured TRHEPD rocking curves and determined the atomic arrangements of the topmost crystal surface [3].

A total reflection high-energy positron diffraction station at the KEK-SPF

A high-intensity mono-energetic positron beam generated by using a linear electron accelerator (linac) provides total reflection high-energy positron diffraction (TRHEPD) researches at Slow Positron Facility (SPF), KEK [1,2]. A pulsed 50-Hz electron beam generated with a dedicated linac (operated at 55 MeV, 0.6 kW) is injected on a Ta converter and causes fast positron-electron pair creation through bremsstrahlung. The positrons showering down on 25 μm-thick W foils, are moderated to thermal energy, and a fraction spontaneously comes out of the foils with an energy of 3 eV owing to the negative work function. The positron converter/moderator assembly is held at an electrostatic voltage of 15 kV for TRHEPD experiment. The emitted positrons are consequently accelerated to 15 keV as they enter the grounded beam-line and are guided by the magnetic field of about 0.015 T to the TRHEPD station. For diffraction experiments, positrons transported by the magnetic field have to be first released into a nonmagnetic region. Since the released positron beam has a large diameter, a brightness-enhancement unit is effectively used to achieve a small-diameter and highly-parallel beam with a sufficient flux. The linac-based positron beam gives about 60 times intensified diffraction pattern from a Si(111)-7x7 reconstructed surface compared to a previous result with a Na-22-based positron beam [3]. An improved signal-to-noise ratio in the obtained pattern due to the intensified beam allowed an observation of clear fractional-order spots in the higher Laue-zones, which had not been observed previously. The much intensified beam with the present system allows adjustment of the sample orientation without accumulating the positron signals. With the brightness enhanced beam, several remarkable results have been obtained efficiently by users of this facility. (Everybody is invited to use KEK Slow Positron Facility through approval of his/her research proposal.)

Structural analyses of the two cysteine desulfurases from Aquifex aeolicus

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer to a wide range of acceptor proteins. IscS degrades L-cysteine into L-alanine and a sulfur atom in a pyridoxal 5'-phosphate (PLP) dependent manner. In this reaction, it is essential for a conserved Lys residue of IscS to form Schiff base (the covalent bonding interaction) with PLP. Recent accumulations of genomic information have revealed that some IscS homologues in archaea and thermophilic bacteria lack this invariant Lys. Here we report the crystal structures of two paralogous cysteine desulfurases, the canonical Aa IscS1 and the invariant Lys lacking Aa IscS2, from Aquifex aeolicus. Aa IscS1/Aa IscS2 were overproduced in E. coli, and purified by heat-treatment and several column chromatography, and crystallized. The structure of Aa IscS1 was determined at 2.00 Å (Rcryst= 19.4% and Rfree = 22.0%), and Aa IscS2 at 2.55 Å (Rcryst= 21.8% and Rfree = 27.0%). Overall structures as well as orientations of the residues in the active site were quite similar to each other. In Aa IscS1 the PLP adduct was anchored in the catalytic pocket of Aa IscS1 by the formation of the aldimine Schiff base with the invariant Lys. Whereas in Aa IscS2 the PLP was not seen in the active pocket, since the catalytic Lys was substituted by Leu. Alternatively, an electron density derived from unknown-small molecule was located in the catalytic site of Aa IscS2. The shape of this electron density was completely different from that of PLP. The Bijvoet difference map calculated from data collected at λ=1.7 Å overlapped with the electron density observed in the active site; the unknown-small molecule probably contains such metals as iron atoms. Furthermore, the ICP-MS analysis demonstrated that as-isolated Aa IscS2 harbored the iron atom in the solution state. More recently we obtained the experimental evidences that non-canonical Aa IscS2 was able to form the binary complex with Aa IscU, which is responsible for a scaffold for the assembly of a nascent Fe-S cluster. Base on the structural/biochemcal results, possible physiological functions of two cysteine desulfrurases will be discussed.