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Thomas M Makris

Publications and source records attributed to Thomas M Makris.

11 recordsLinked to original sources
PubMed2006

Cytochrome p450 compound I.

Cytochrome P450 enzymes (P450s) comprise a large class of enzymes that effect numerous oxidations in nature. The active oxidants in P450s are thought to be iron(IV)-oxo porphyrin radical cations termed Compounds I, and these intermediates have been sought since the discovery of P450s 40 years ago. We report formation of the Compound I derivative of a P450 enzyme by laser flash photolysis oxidation of the corresponding Compound II species, an iron(IV)-oxo neutral porphyrin intermediate. The Compound II derivative in turn was produced by oxidation of the P450 with peroxynitrite, which effected a net one-electron, oxo-transfer reaction to the iron(III) atom of the resting enzyme. For the P450 studied in this work, CYP119 from the thermophile Sulfolobus solfactaricus, the P450 Compound II derivative was stable for seconds at ambient temperature, and the Compound I transient decayed with a lifetime of ca. 200 ms.

Archaeal Proteins↗
PubMed2006

The status of high-valent metal oxo complexes in the P450 cytochromes.

The oxidative prowess of the P450 cytochromes in physiological reactions is attributed to the production of a high-valent iron-oxo complex, or Compound I intermediate, in the reaction cycle. Despite many years of study, however, the full electronic description of this fleeting intermediate still remains an active area of study. In this manuscript, the current status of the isolation and characterization of the P450 oxo-Fe(IV) is examined and compared to analogous states in related heme enzymes. In addition, the utilization of cofactor exchange to stabilize high-valent oxo-states in the P450 is addressed. Structural and spectroscopic studies on manganese reconstituted P450, and its corresponding oxo-complex, are presented.

Cytochrome P-450 Enzyme System↗
PubMed2005

Thirty years of microbial P450 monooxygenase research: peroxo-heme intermediates--the central bus station in heme oxygenase catalysis.

Oxygen has always been recognized as an essential element of many life forms, initially through its role as a terminal electron acceptor for the energy-generating pathways of oxidative phosphorylation. In 1955, Hayaishi et al. [Mechanism of the pyrocatechase reaction, J. Am. Chem. Soc. 77 (1955) 5450-5451] presented the most important discovery that changed this simplistic view of how Nature uses atmospheric dioxygen. His discovery, the naming and mechanistic understanding of the first "oxygenase" enzyme, has provided a wonderful opportunity and scientific impetus for four decades of researchers. This volume provides an opportunity to recognize the breakthroughs of the "Hayaishi School." Notable have been the prolific contributions of Professor Ishimura et al. [Oxygen and life. Oxygenases, Oxidases and Lipid Mediators, International Congress Series, Elsevier, Amsterdam, 2002], a first-generation Hayaishi product, to characterization of the cytochrome P450 monooxygenases.

Bacterial Proteins↗
PubMed2005

The influence of substrate on the spectral properties of oxyferrous wild-type and T252A cytochrome P450-CAM.

To probe whether the nature of the substrate can directly influence the spectral properties of oxyferrous cytochrome P450-CAM, the complex has been investigated in the absence and in the presence of the natural substrate (1R)-camphor (camphor) and of several camphor analogs. The oxyferrous complex of T252A P450-CAM, a mutant lacking the hydroxyl group that forms a hydrogen bond to the heme iron-coordinated dioxygen, has also been studied to gauge the influence of this hydrogen bond. UV-visible absorption and magnetic circular dichroism (MCD) spectra of these oxyferrous adducts prepared and stabilized at -40 degrees C in 60% (v/v) ethylene glycol are generally similar, exhibiting absorption bands at approximately 355, approximately 420, approximately 554, and approximately 585 nm (shoulder) and a characteristic MCD trough at approximately 585 nm. The MCD spectrum of camphor-bound oxyferrous P450-CAM is similar to that of the substrate-free oxyferrous enzyme, but the spectrum of the oxyferrous enzyme differs detectably in the presence of substrate analogs. The spectra of the oxyferrous T252A mutant and wild-type enzyme are overall similar except for Soret band position blue shifts by 2-6 nm for the mutant. 5-Methylenylcamphor (epoxidation substrate) appears to have an anomalous binding mode for the mutant compared with that for the wild-type enzyme. The present results indicate that the structures of the camphor analogs can sensitively influence the physical (spectroscopic) properties of the P450 dioxygen complex and could also affect its reactivity. The ability of substrate to modulate the reactivity of P450 intermediates could be a relevant factor in explaining the remarkable diversity of reactions catalyzed by the enzyme.

Binding Sites↗
PubMed2005

A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803.

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.

Bacterial Proteins↗
PubMed2003

Resonance Raman spectroscopic studies of hydroperoxo-myoglobin at cryogenic temperatures.

In agreement with previous reports (Gasyna, Z. FEBS Lett. 1979, 106, 213-218 and Leibl, W.; Nitschke, W.; Huettermann, J. Biochim. Biophys. Acta 1986, 870, 20-30) radiolytically reduced samples of oxygenated myoglobin at cryogenic temperatures have been shown by optical absorption and EPR studies to produce directly the peroxo-bound myoglobin at 77 K. Annealing to temperatures near 185 K induces proton transfer, resulting in the formation of the hydroperoxo heme derivative. Resonance Raman studies of the annealed samples has permitted, for the first time, the direct observation of the key nu(Fe-O) stretching mode of the physiologically important Fe-OOH fragment of this ubiquitous intermediate. The assignment of this mode to a feature appearing at 617 cm(-1) is strongly supported by documentation of a 25 cm(-1) shift to lower energy upon substitution with (18)O(2) and by a 5 cm(-1) shift to lower energy for samples prepared in solutions of deuterated solvent.

Animals↗
PubMed2003

Epoxidation of olefins by hydroperoxo-ferric cytochrome P450.

The T252A mutant of cytochrome P450cam is unable to form the oxoferryl "active oxygen" intermediate, as judged by its inability to hydroxylate its normal substrate, camphor. In the present study, we demonstrate that T252A P450cam is nonetheless able to epoxidize olefins, due to the action of a second oxidant. However, as shown in earlier radiolytic studies and by the ability of T252A to reduce dioxygen to hydrogen peroxide, the mutant retains the ability to form the hydroperoxo-ferric reaction cycle intermediate. The present results provide strong evidence that hydroperoxo-ferric P450 can serve as a second electrophilic oxidant capable of olefin epoxidation.

Alkanes↗
PubMed2002

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis.

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.

Electron Spin Resonance Spectroscopy↗
PubMed2002

Mechanistic enzymology of oxygen activation by the cytochromes P450.

The P450 cytochromes represent a universal class of heme-monooxygenases. The detailed mechanistic understanding of their oxidative prowess is a critical theme in the studies of metabolism of a wide range of organic compounds including xenobiotics. Integral to the O2 bond cleavage mechanism by P450 is the enzyme's concerted use of protein and solvent-mediated proton transfer events to transform reduced dioxygen to a species capable of oxidative chemistry. To this end, a wide range of kinetic, structural, and mutagenesis data has been accrued. A critical role of conserved acid-alcohol residues in the P450 distal pocket, as well as stabilized waters, enables the enzyme to catalyze effective monooxygenation chemistry. In this review, we discuss the detailed mechanism of P450 dioxygen scission utilizing the CYP101 hydroxylation of camphor as a model system. The application of low-temperature radiolytic techniques has enabled a structural and spectroscopic analysis of the nature of critical intermediate states in the reaction.

Animals↗