Examination of the role of methylenetetrahydrofolate reductase in incorporation of methyltetrahydrofolate into cellular metabolism

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154338/1/fsb2002001005.pd

Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa

The monooxygenase, p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) has been isolated and purified from Pseudomonas aeruginosa. The reaction catalysed is linked to the pathways for degradation of aromatic compounds by microorganisms. The enzyme has been quantitatively characterizd in this paper for use in the mechanistic analysis of the protein by site-directed mutagenesis. This can be achieved when the results presented are used in combination with the information on the sequence and structure of the gene for this protein and the high-resolution crystallographic data for the protein from P. fluorescens. The protein is a dimer of identical sub-units in solution, and has one FAD per polypeptide with a monomeric molecular weight of 45 000. A full steady-state kinetic analysis was carried out at the optimum pH (8.0). A Vmax of 3750 min-1 at 25[deg]C was calculated, and the enzyme has a concerted-substitution mechanism, involving the substrates, NADPH, oxygen, and p-hydrobenzoate. Extensive analyses of the reactions of reduced enzyme with oxygen were carried out. The quality of the data obtained confirmed the mechanisms of these reactions as proposed earlier by the authors for the enzyme from P. fluorescens. It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis. Thus, transient state kinetic analysis is an excellent tool to examine the role of amino acid residues in catalysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27633/1/0000009.pd

Structure and mechanism of the iron‐sulfur flavoprotein phthalate dioxygenase reductase

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154520/1/fsb2009014006.pd

Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450LMredComplex I-->Complex II-->P-450LMox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome is added. The latter protein does not appear to function as an electron carrier in this process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21753/1/0000147.pd

The FAD Cofactor of RebC Shifts to an IN Conformation upon Flavin Reduction†,‡

RebC is a putative flavin hydroxylase functioning together with RebP to carry out a key step in the biosynthesis of rebeccamycin. To probe the mechanism of flavin-based chemistry in RebC, we solved the structure of RebC with reduced flavin. Upon flavin reduction, the RebC crystal undergoes a change in its unit cell dimension concurrent with a 5 Å movement of the isoalloxazine ring, positioning the flavin ring adjacent to the substrate-binding pocket. Additionally, a disordered helix becomes ordered upon flavin reduction, closing off one side of the substrate-binding pocket. This structure, along with previously reported structures, increases our understanding of the RebC enzyme mechanism, indicating that either the reduction of the flavin itself or binding of substrate is sufficient to drive major conformational changes in RebC to generate a closed active site. Our finding that flavin reduction seals the active site such that substrate cannot enter suggests that our reduced flavin RebC structure is off-pathway and that substrate binding is likely to precede flavin reduction during catalysis. Along with kinetic data presented here, these structures suggest that the first cycle of catalysis in RebC may resemble that of p-hydroxybenzoate hydroxylase, with substrate binding promoting flavin reduction

Oxidative Protein Folding Is Driven by the Electron Transport System

AbstractDisulfide bond formation is catalyzed in vivo by DsbA and DsbB. Here we reconstitute this oxidative folding system using purified components. We have found the sources of oxidative power for protein folding and show how disulfide bond formation is linked to cellular metabolism. We find that disulfide bond formation and the electron transport chain are directly coupled. DsbB uses quinones as electron acceptors, allowing various choices for electron transport to support disulfide bond formation. Electrons flow via cytochrome bo oxidase to oxygen under aerobic conditions or via cytochrome bd oxidase under partially anaerobic conditions. Under truly anaerobic conditions, menaquinone shuttles electrons to alternate final electron acceptors such as fumarate. This flexibility reflects the vital nature of the disulfide catalytic system

XAS investigation of the Fe sites in phthalate dioxygenase

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27899/1/0000319.pd

Properties of partially purified liver microsomal cytochrome P-450: Acceptance of two electrons during anaerobic titration

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22425/1/0000875.pd

Structural characterization of the mononuclear iron site in Pseudomonas cepacia phthalate DB01 dioxygenase using X-ray absorption spectroscopy

 Phthalate dioxygenase (PDO) from Pseudomonas cepacia contains a Rieske-like [2Fe-2S] cluster and a mononuclear non-heme Fe(II) site. The mononuclear iron can be replaced by a variety of divalent metal ions, although only Fe(II) permits catalytic activity. We used X-ray absorption spectroscopy to characterize the structural properties of the mononuclear iron site and to follow the structural changes in this site as a function both of Rieske site oxidation state and of phthalate binding. Data for the mononuclear site have been measured directly for PDO substituted with Co or Zn in the mononuclear site, and by difference for the native 3-Fe protein. The mononuclear site was modeled well by low Z-ligation (oxygen or nitrogen) and showed no evidence for high-Z ligands (e.g., sulfur). The relatively short average first shell bond lengths and the absence of significant outer shell scattering suggest that the mononuclear site has several oxygen ligands. With Zn in the mononuclear site, the average bond length (2.00 Å) suggests a 5-coordinate site under all conditions. In contrast, the Co- or Fe-containing mononuclear site appeared to be 6-coordinate and changed to 5-coordinate when substrate was bound, since the first shell bond length changed from 2.08 to 2.02 Å (Co) or 2.10 to 2.06 Å (Fe). The implications of these findings for the PDO mechanism are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42321/1/775-1-1-24_60010024.pd