On-The-Fly Observing System of the Nobeyama 45-m and ASTE 10-m Telescopes

We have developed spectral line On-The-Fly (OTF) observing mode for the Nobeyama Radio Observatory 45-m and the Atacama Submillimeter Telescope Experiment 10-m telescopes. Sets of digital autocorrelation spectrometers are available for OTF with heterodyne receivers mounted on the telescopes, including the focal-plane 5 x 5 array receiver, BEARS, on the 45-m. During OTF observations, the antenna is continuously driven to cover the mapped region rapidly, resulting in high observing efficiency and accuracy. Pointing of the antenna and readouts from the spectrometer are recorded as fast as 0.1 second. In this paper we report improvements made on software and instruments, requirements and optimization of observing parameters, data reduction process, and verification of the system. It is confirmed that, using optimal parameters, the OTF is about twice as efficient as conventional position-switch observing method.Comment: 11 pages, 13 figures, accepted for publication in PAS

Fundamental electron-transfer and proton-coupled electron-transfer properties of Ru(iv)-oxo complexes

Isolation and characterisation of Ru-IV(O) complexes were accomplished to investigate their fundamental electron transfer (ET) and proton-coupled ET (PCET) properties. Reorganisation energies (lambda) in electron transfer (ET) and proton-coupled ET (PCET) from electron donors to the isolated Ru-IV(O) complexes have been determined for the first time to be in the range of 1.70-1.88 eV (ET) and 1.20-1.26 eV (PCET). It was suggested that the reduction of the lambda values of PCET in comparison with those of ET should be due to the smaller structural change in PCET than that in ET on the basis of DFT calculations on 1 and 1e(-)-reduced 1 in the absence and presence of TFA, respectively. In addition, the smaller lambda values for the Ru-IV(O) complexes than those reported for Fe-IV(O) and Mn-IV(O) complexes should be due to the lack of participation of d(sigma) orbitals in the ET and PCET reactions. This is the first example to evaluate fundamental ET and PCET properties of Ru-IV(O) complexes leading to further understanding of their reactivity in oxidation reactions

Brown adipose tissue dysfunction promotes heart failure via a trimethylamine N-oxide-dependent mechanism.

Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.We thank Kaori Yoshida, Keiko Uchiyama, Satomi Kawai, Naomi Hatanaka, Yoko Sawaguchi, Runa Washio, Takako Ichihashi, Nanako Koike, Keiko Uchiyama, Masaaki Nameta (Niigata University), Kaori Igarashi, Kaori Saitoh, Keiko Endo, Hiroko Maki, Ayano Ueno, Maki Ohishi, Sanae Yamanaka, Noriko Kagata (Keio University) for their excellent technical assistance, C. Ronald Kahn (Joslin Diabetes Center and Harvard Medical School) for providing the BAT cell line, Evan Rosen (Harvard Medical School) for providing us Ucp-Cre mice, Kosuke Morikawa (Kyoto University), Tomitake Tsukihara (University of Hyogo) and Shinya Yoshikawa (University of Hyogo) for their professional opinions and suggestions. Tis work was supported by a Grant-in-Aid for Scientifc Research (A) (20H00533) from MEXT, AMED under Grant Numbers JP20ek0210114, and AMED-CREST under Grant Number JP20gm1110012, and Moonshot Research and Development Program (21zf0127003s0201), MEXT Supported Program for the Strategic Research Foundation at Private Universities Japan, Private University Research Branding Project, and Leading Initiative for Excellent Young Researchers, and grants from the Takeda Medical Research Foundation, the Vehicle Racing Commemorative Foundation, Ono Medical Research Foundation, and the Suzuken Memorial Foundation (to T.M.). Support was also provided by a Grants-in-Aid for Young Scientists (Start-up) (26893080), and grants from the Uehara Memorial Foundation, Kowa Life Science Foundation, Manpei Suzuki Diabetes Foundation, SENSHIN Medical Research Foundation, ONO Medical Research Foundation, Tsukada Grant for Niigata University Medical Research, Te Nakajima Foundation, SUZUKEN memorial foundation, HOKUTO Corporation, Mochida Memorial Foundation for Medical & Pharmaceutical Research, Grants-in-Aid for Encouragement of Young Scientists (A) (16H06244), Daiichi Sankyo Foundation of Life Science, AMED Project for Elucidating and Controlling Mechanisms of Aging and Longevity under Grant Number JP17gm5010002, JP18gm5010002, JP19gm5010002, JP20gm5010002, JP21gm5010002, Astellas Foundation for Research on Metabolic Disorders, Research grant from Naito Foundation, Te Japan Geriatrics Society (to I.S.); by a Grant-in-Aid for Scientifc Research (C) (19K08974), Yujin Memorial Grant, Sakakibara Memorial Research Grant from Te Japan Research Promotion Society for Cardiovascular Diseases, TERUMO Life Science Foundation, Kanae Foundation (to Y.Y.), JST ERATO (JPMJER1902), AMED-CREST (JP20gm1010009), the Takeda Science Foundation, the Food Science Institute Foundation (to S.F.), and by a grant from Bourbon (to T.M., I.S. and Y.Y.).S

Active Site Engineering of Copper-Containing Electron Transfer Proteins

Cupredoxins arc electron transfer (ET) proteins which possess type I (Tl) copper sites. A TI copper ion is equatorially coordinated by the thiolate sulfur of a Cys and the imidazole nitrogens of two His residues, along with usually an axially coordinating thioether sulfur of a Met [azurin (Az) possess a second axial interaction with a backbone carbonyl oxygen]. Thr~e of these ligands (Cys, a His and Met) are found on a C-terminal loop which links two of the strands of the cupredoxin p-barrel scaffold. The length and sequence of this Tl copper-binding loop varies. The shOltest known Tl binding loop (that of amicyanin, Ami) has been introduced into three different cupredoxin scaffolds. All of the . loopcontraction variants possess copper centres with authentic TI properties and are redox active. The Cu(lI) and Co(II) sites experience only small structural alterations upon loop contraction with the largest changes in the Az variant (AzAmi) which can be ascribed to the removal of a hydrogen bond to the coordinating thiolate sulfur of the Cys ligand. In all cases loop-contraction leads to an increase in the pKa of the His ligand found on the loop in the reduced proteins, and in the pseudoazurin (Paz) and plastocyanin (Pc) variants the values are almost identical to that of Ami (~6.7). Thus in Paz, Pc and Ami the length of this loop tunes the pKa of the His ligand. In the AzAmi variant the pKa is 5.5 which is considerably higher than the estimated value for Az « 2) and other controlling factors, along with loop length, are involved. The reduction potentials (EmS) of the loop-contraction variants are all lower than those of the wild type (WT) proteins by ~ 30-60 mV and thus this property of a Tl copper site is finetuned by the C-tenninalloop. The electron self-exchange (ESE) rate constant (kESE) of Paz is diminished significantly by the introduction of a shorter loop. However, in PcAmi only a 2-fold decrease is observed and in AzAmi there is no effect, and thus in these two cupredoxins loop contraction does not signi~cantly influence ET reactivity. Loop-contraction provides an active site environment in all of the cup.re~oxins which is preferable for Cu(ll), whereas previous loop elongation experiments always favoure..d the cuprous site. Thus the ligand-containing loop plays an important role in tuning the entatic nature ora TI copper centre. The thiolate sulfur of the Cys ligand in Az is hydrogen bonded from the backbone amides of .Asn47 and Phe114. One of these interactions has been removed in the Phel14Pro variant. A unique . I'~ pectr~s~opj~=..featur~ ,of A.z is-the position of.the S(Cys)~Cu(Il),.ligand-to-metalcharge-transfer . - .. .' ~, . . .' . . (LMCT) band in its UVNis spectrum (~ 630 nm) which is shifted to 599 nm in the Phel14Pro variant, although a site with classic Tl properties is maintained. Shorter CU(Il)-SO(Metl21) and longer Cu(lI}O( Gly45) distances are found at the active site in the crystal structure of the variant compared to WT Az. The copper centre of Phel14Pro Az is more like those of Pc, Ami and Paz than the trigonal bipyramidal arrangement found in Az. The Phel14Pro mutation results in an 80 mV decrease in Em and an order of magnitude smaller kESE value. The influence of this mutation on Em is due to a number of structural effects ofthe mutation, with removal of the hydrogen bond probably most significant. Comparison of the active site structures of Cu(ll) and Cu(I) Phe114Pro· Az indicate larger changes upon redox interconversion than those in the WT protein which increases the reorganization energy and results in slower ET. The axial ligand at Tl copper sites is not conserved. In most cases a weakly coordinated thioether sulfur from a Met [Cu(II)-S5 ~ 2.6-3.3 A] is found in the axial position as in Pc, Paz, Az and Ami. A strong axial bond [Cu(II)-Otl of ~ 2.2 A] is sometimes provided by a Gln.[as in the stellacyanins (STCs)] and the axial ligand can be absent (a Val, Leu or Phe in the axial position) as in ceruloplasmin, FeOp, fungal laccases and some plantacyanins (PLTs). Cucumber basic protein (CBP) is a PLT which has a relatively short Cu(II)-S5(Met89) axial bond (2.6 A). The Met89Gln variant of CBP has a kE?E' a measure of intrinsic ET reactivity, ~ 7 times lower than that of the WT protein. The Met89Vai mutation to CBP results in a 2-fold increase in kESE' As the axial interaction decreases from strong Oel of Gin to relatively w.eak S5 of Met to no ligand (Val), ESE reactivity is enhanced by - 1 order of magnitude whilst Em increases by - 350 mY. The variable coordination position at this ubiquitous ET site provides a mechanism for tuning the driving force to optimize ET with the correct partner without significantly compromising intrinsic reactivity. The enhanced reactivity of a three-coordinate Tl copper site will facilitate intramolecular ET in fungallaccases and Fet3p. The phytocyanins form a sub-family of the cupredoxins and are made up of the STCs, PLTs and uc1acyanins. All of the phytocyanins exhibit an alkaline transition which results in the S(Cys)~Cu(II) LMCT band shifting - 20 nm to higher energy at elevated pH (pKa - 10). The alkaline transition influences all of the coordinating residues with the Cys ligand most affected. The exact cause of alkaline transition is not known, although deprotonation of a group close to the active site must be involved, and the side chain of the axial Gin ligand has been suggested as the trigger for this effect in the STCs. The influe!lce of pH on the spectrosco~ic properties of WT CBP and the Met89Gln and Met89Vai axial ligand variants has been studied. The alkaline transition has a similar influence on . the visible spectrum in all three proteins although the pKa value in Met89Vai CBP is smaller (8.9) than for the other two proteins (- 9.7). Thus the axial ligand is not the cause ofthe alkaline transition. The surface exposed Met16 residue of Paz is situated close to the His81 ligand in the centre of the protein's hydrophobic patch. To study the importance of Met'i6, and to attempt to introduce a 1t-1t. interaction with the imidazole ring of His81, the Met16Phe and Met16Trp variants have been prepared and characterized. NMR studies indicate that the introduced aromatic groups are oriented parallel to the imidazole ring of His8l. UVNis, EPR and paramagnetic IH NMR spectra of the Cu(II) variants highlight very similar active site structures in the two mutants which are less tetragonally distorted than in the WT protein. The pKa value for the His81 ligand in the Cu(I) proteins decreases from 4.9 in WT Paz to 4.5 and 4.1 in Metl6Phe and Metl6Trp Paz respectively, indicating that 1t-1t contact with the introduced aromatic ring stabilizes the Cu-N51 (His81) interaction. The enhanced rigidity at the active site may contribute to decr~ased reorganization energies in the variants resulting in - 2-fold and - 3-fold larger kESE values in Met16Phe alid Metl6Trp Paz respectively. These mutations could also contribute to tl~e increased kESE values by facilitating homo-dimer formation: The Metl6Phe and Metl6Trp mutations give rise to approximately 40-60 mV increases in the Em of Paz. The physiological function of Paz is donation of electrons to nitrite reductace (NiR) and the influence of these mutations on Em result in a decreased driving force for this ET reaction and smaller kC31 are found. The Km for the reaction with NiR is - 2-fold larger for the Met16Phe variant whilst similar values are found for Met 16Trp Paz and the WT protein. Introduction of a 1t-1t interaction at the active site of Paz leads to subtle structural changes but has little effect on the interaction with the physiological ET partner.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Investigating the cause of the alkaline transition in phytocyanins

The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His2Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic 1H NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pKa of ~10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pKa for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pKa for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition