131 research outputs found
Control of substrate access to the active site in methane monooxygenase
Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH–MMOB (hereafter termed H–B) complex. Here we remedy this deficiency by providing a crystal structure of H–B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α[subscript 2]β[subscript 2] interface of α[subscript 2]β[subscript 2]γ[subscript 2] MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle.National Institute of General Medical Sciences (U.S.) (Grant GM 32114
A Transition State Analogue for the Oxidation of Binuclear Palladium(II) to Binuclear Palladium(III) Complexes
Failure of purified alcoholic hyalin to induce lymphocyte transformation in acute alcoholic hepatitis
Structure, electronic configuration, and Mössbauer spectral parameters of an antiferromagnetic Fe2-peroxo intermediate of methane monooxygenase
Identification of Protein-Bound Dinitrosyl Iron Complexes by Nuclear Resonance Vibrational Spectroscopy
We have applied [superscript 57]Fe nuclear resonance vibrational spectroscopy (NRVS) to identify protein-bound dinitrosyl iron complexes. Intense NRVS peaks due to vibrations of the N−Fe−N unit can be observed between 500 and 700 cm[superscript −1] and are diagnostic indicators of the type of iron dinitrosyl species present. NRVS spectra for four iron dinitrosyl model compounds are presented and used as benchmarks for the identification of species formed in the reaction of Pyrococcus furiosus ferredoxin D14C with nitric oxide.National Science Foundation (Grant Number CHE-0611944)National Institute of General Medical Sciences (U.S.). Interdepartmental Training (Grant Number T32 GM08334)National Institute of General Medical Sciences (U.S.). Postdoctural Fellowship (1 F32 GM082031-02
Mechanistic Studies of Reactions of Peroxodiiron(III) Intermediates in T201 Variants of Toluene/o-Xylene Monooxygenase Hydroxylase
Site-directed mutagenesis studies of a strictly conserved T201 residue in the active site of toluene/o-xylene monooxygenase hydroxylase (ToMOH) revealed that a single mutation can facilitate kinetic isolation of two distinctive peroxodiiron(III) species, designated T201peroxo and ToMOHperoxo, during dioxygen activation. Previously, we characterized both oxygenated intermediates by UV–vis and Mössbauer spectroscopy, proposed structures from DFT and QM/MM computational studies, and elucidated chemical steps involved in dioxygen activation through the kinetic studies of T201peroxo formation. In this study, we investigate the kinetics of T201peroxo decay to explore the reaction mechanism of the oxygenated intermediates following O2 activation. The decay rates of T201peroxo were monitored in the absence and presence of external (phenol) or internal (tryptophan residue in an I100W variant) substrates under pre-steady-state conditions. Three possible reaction models for the formation and decay of T201peroxo were evaluated, and the results demonstrate that this species is on the pathway of arene oxidation and appears to be in equilibrium with ToMOHperoxo.National Institute of General Medical Sciences (U.S.) (GM032134
Inactivation of the Enveloped Bacteriophage ø6 by Butylated Hydroxytoluene and Butylated Hydroxyanisole
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