Infrared spectroscopy has been used to examine the oxidized and CO-inhibited forms of Fe-only hydrogenase I from Clostridium pasteurianum. For the oxidized enzyme, five bands are detected in the infrared spectral region between 2100 and 1800 cm(-1). The pattern of infrared bands is consistent with the presence of two terminally coordinated carbon monoxide molecules, two terminally coordinated cyanide molecules, and one bridging carbon monoxide molecule, ligated to the Fe atoms of the active site [2Fe] subcluster. Infrared spectra of the carbon monoxide-inhibited state, prepared using both natural abundance CO and 13CO, indicate that the two terminally coordinated CO ligands that are intrinsic to the enzyme are coordinated to different Fe atoms of the active site [2Fe] subcluster. Irradiation of the CO-inhibited state at cryogenic temperatures gives rise to two species with dramatically different infrared spectra. The first species has an infrared spectrum identical to the spectrum of the oxidized enzyme, and can be assigned as arising from the photolysis of the exogenous CO from the active site. This species, which has been observed in X-ray crystallographic measurements [Lemon, B. J., and Peters, J. W. (2000) J. Am. Chem. Soc. 122, 3793], decays above 150 K. The second light-induced species decays above 80 K and is characterized by loss of the infrared band associated with the Fe bridging CO at 1809 cm(-1). Potential models for the second photolysis event are discussed.
Intensive crop production systems worldwide, particularly in China, rely heavily on nitrogen (N) fertilization, but left more than 50% of fertilizer N in the environment. Nitrogen (over) fertilization and atmospheric N deposition induce soil acidification, which is neu-
Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of hand, foot, and mouth disease (HFMD), which is prevalent in Asia. Thus far, there are no prophylactic or therapeutic measures against HFMD. The 3C proteases from EV71 and CVA16 play important roles in viral replication and are therefore ideal drug targets. By using biochemical, mutational, and structural approaches, we broadly characterized both proteases. A series of high-resolution structures of the free or substrate-bound enzymes were solved. These structures, together with our cleavage specificity assay, well explain the marked substrate preferences of both proteases for particular P4, P1, and P1 residue types, as well as the relative malleability of the P2 amino acid. More importantly, the complex structures of EV71 and CVA16 3Cs with rupintrivir, a specific human rhinovirus (HRV) 3C protease inhibitor, were solved. These structures reveal a half-closed S2 subsite and a size-reduced S1 subsite that limit the access of the P1 group of rupintrivir to both enzymes, explaining the reported low inhibition activity of the compound toward EV71 and CVA16. In conclusion, the detailed characterization of both proteases in this study could direct us to a proposal for rational design of EV71/CVA16 3C inhibitors.
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