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

An Integrated Proteomics/Transcriptomics Approach Points to Oxygen as the Main Electron Sink for Methanol Metabolism in Methylotenera mobilis▿†

Methylotenera species, unlike their close relatives in the genera Methylophilus, Methylobacillus, and Methylovorus, neither exhibit the activity of methanol dehydrogenase nor possess mxaFI genes encoding this enzyme, yet they are able to grow on methanol. In this work, we integrated a genome-wide proteomics approach, shotgun proteomics, and a genome-wide transcriptomics approach, shotgun transcriptome sequencing (RNA-seq), of Methylotenera mobilis JLW8 to identify genes and enzymes potentially involved in methanol oxidation, with special attention to alternative nitrogen sources, to address the question of whether nitrate could play a role as an electron acceptor in place of oxygen. Both proteomics and transcriptomics identified a limited number of genes and enzymes specifically responding to methanol. This set includes genes involved in oxidative stress response systems, a number of oxidoreductases, including XoxF-type alcohol dehydrogenases, a type II secretion system, and proteins without a predicted function. Nitrate stimulated expression of some genes in assimilatory nitrate reduction and denitrification pathways, while ammonium downregulated some of the nitrogen metabolism genes. However, none of these genes appeared to respond to methanol, which suggests that oxygen may be the main electron sink during growth on methanol. This study identifies initial targets for future focused physiological studies, including mutant analysis, which will provide further details into this novel process

Multiple Roles of Component Proteins in Bacterial Multicomponent Monooxygenases: Phenol Hydroxylase and Toluene/ o

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H2O2) generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was 50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H2O2 formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.National Institute of General Medical Sciences (U.S.) (grant GM032134)National Institutes of Health (U.S.) (Interdepartmental Biotechnology Training Grant T32 GM08334)CEINGE - Biotecnologie Avanzate (Italy

Intermediate P* from Soluble Methane Monooxygenase Contains a Diferrous Cluster

During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the Fe(II)Fe(II) state (H(red)). O(2) binds rapidly at a site away from the cluster to form the Fe(II)Fe(II) intermediate O, which converts to an Fe(III)Fe(III)-peroxo intermediate P and finally to the Fe(IV)Fe(IV) intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of H(red) and O is lost. This was interpreted as signaling oxidation of the cluster, but low accumulation of P* prevented further characterization. In this study, three methods to directly detect and trap P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of H(red) and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the Fe(II)Fe(II) state. Thus, the loss of the g = 16 EPR results from a change of the electronic structure of the Fe(II)Fe(II) center rather than oxidation. The similarity of the optical and Mössbauer spectra of H(red), O, and P* suggest that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O(2) to be activated for hydrocarbon oxidation