Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

Background: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration ( Scope of review: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. Major conclusions: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. General significance: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules

Recent progress in experimental studies on the catalytic mechanism of cytochrome c oxidase

Cytochrome c oxidase (CcO) reduces molecular oxygen (O2) to water, coupled with a proton pump from the N-side to the P-side, by receiving four electrons sequentially from the P-side to the O2-reduction site—including Fea3 and CuB—via the two low potential metal sites; CuA and Fea. The catalytic cycle includes six intermediates as follows, R (Fea32+, CuB1+, Tyr244OH), A (Fea32+-O2, CuB1+, Tyr244OH), Pm (Fea34+ = O2−, CuB2+-OH−, Tyr244O•), F (Fea34+ = O2−, CuB2+-OH-, Tyr244OH), O (Fea33+-OH-, CuB2+-OH−, Tyr244OH), and E (Fea33+-OH-, CuB1+-H2O, Tyr244OH). CcO has three proton conducting pathways, D, K, and H. The D and K pathways connect the N-side surface with the O2-reduction site, while the H-pathway is located across the protein from the N-side to the P-side. The proton pump is driven by electrostatic interactions between the protons to be pumped and the net positive charges created during the O2 reduction. Two different proton pump proposals, each including either the D-pathway or H-pathway as the proton pumping site, were proposed approximately 30 years ago and continue to be under serious debate. In our view, the progress in understanding the reaction mechanism of CcO has been critically rate-limited by the resolution of its X-ray crystallographic structure. The improvement of the resolutions of the oxidized/reduced bovine CcO up to 1.5/1.6 Å resolution in 2016 provided a breakthrough in the understanding of the reaction mechanism of CcO. In this review, experimental studies on the reaction mechanism of CcO before the appearance of the 1.5/1.6 Å resolution X-ray structures are summarized as a background description. Following the summary, we will review the recent (since 2016) experimental findings which have significantly improved our understanding of the reaction mechanism of CcO including: 1) redox coupled structural changes of bovine CcO; 2) X-ray structures of all six intermediates; 3) spectroscopic findings on the intermediate species including the Tyr244 radical in the Pm form, a peroxide-bound form between the A and Pm forms, and Fr, a one-electron reduced F-form; 4) time resolved X-ray structural changes during the photolysis of CO-bound fully reduced CcO using XFEL; 5) a simulation analysis for the Pm→Pr→F transition

Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8

Study of Sumida River, Part-1; Its COD and EC characteristics from data collected in spring, 2021

The Sumida River, a typical urban river in Tokyo, has been recognized as a site for fostering Tokyo’s urbanlife that serves as a venue of leisure and relaxation for its residents. As the Teikyo University of Science(TUS) is located near the Sumida River, students spend a lot of time nearby. Clarifying the characteristics ofSumida River, and teaching it to students, are important not only for enhancing environmental awarenessamong students, but also local environment conservation. In this study, chemical oxygen demand (COD)and electric conductivity (EC) data, major sources of contamination, and the characteristics of the river wereanalyzed and interpreted as follows. 1) Although our COD and EC data showed that Sumida River wasaffected by tidal movement, the river water in our research area (from Otakebashi Bridge to Senju-OhashiBridge) might exhibit backward and forward movements, and it was slightly affected by sea water from theTokyo Bay. 2) Major chemical contaminant sources of investigated area were determined to be the Miyagiwastewater purification facility (WPF) and the Mikawajima WPF at the upstream and downstream sides ofthe TUS, respectively. 3) We presume a two-fraction zone in the Sumida River from our analytical resultsand public data, including a "high COD zone" with an upstream stagnant zone upper the Shirahige Bridge,and a "low COD zone" with a downstream flush zone under the Shirahige Bridge. Raising environmentalawareness concerning the urban river among its residents presumably might contribute to maintaining itsclean and safe environment. We came to conclusion that the importance of educating the citizens aboutkeeping urban rivers environmentally clean and safe for the future generations

Study of Sumida River, Part 2: Analysis of Identification of COD and EC Characteristics in Fall 2021

The Sumida River streamside makes up a part of the Kitasenjyu campus at Teikyo University of Science(TUS). It is essential for university students and faculty members to learn its environmental and chemical characteristics from the viewpoint of environmental education. A water analysis of the Sumida River was conducted in the fall season (Fall 2021) at 10 locations around the Kitasenjyu campus, along with a 24-hour continuous survey in front of the No. 7 building. The fall analysis was performed using the same procedure as the previous analysis in spring (Spring 2021). The conclusions we reached are as follows: 1) Discharge water from the Mikawagima wastewater purification facility (WPF) was found to be the primary source of chemical contamination in the study area, causing an increase in COD (chemical oxygen demand). Upstream, Miyagi WPF was presumed to be the primary source of the chemical contamination. 2) The discharged high COD water from these WPFs was diffused by the tidal movement of Tokyo Bay and then was homogenized in the study area. 3) From our survey on the streaming motion of the Sumida River and public COD data from the Tokyo metropolitan government, we redefined the boundary between high and low COD zones set under the Agastuma Bridge. 4) Urethane foam with photocatalysis material of TiO2 was synthesized as a novel wastewater treatment material. As it displayed good decomposition characteristics oforganic material in tested water, porous and robust materials with TiO2 for continuous outdoor use should be investigated to achieve practical applications shortly

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