Biomedical subjects

John D Lipscomb

Publications and source records attributed to John D Lipscomb.

24 records · Page 2Linked to original sources

PubMed2003

Modulation of substrate binding to naphthalene 1,2-dioxygenase by rieske cluster reduction/oxidation.

The active site of the oxygenase component of naphthalene 1,2-dioxygenase (NDO) contains a Rieske Fe-S cluster and a mononuclear non-heme iron, which are contributed by different alpha-subunits in the (alphabeta)(3) structure. The enzyme catalyzes cis-dihydroxylation of aromatic substrates in addition to numerous other adventitious oxidation reactions. High-resolution Mims (2)H-ENDOR spectra have been recorded for the NO-ferrous center of NDO bound with d(8)-naphthalene and d(2)-naphthalene; spectra were collected for the enzyme with the Rieske diiron center both in its oxidized and in its reduced states. A sharp quartet ENDOR pattern from a nearby deuteron of substrate was detected for each substrate. Examination of the sample prepared with 1,4-dideutero-naphthalene shows that the signal arises from D1. The ENDOR data place D1 at a distance of ca. 4.4 A from the mononuclear Fe and with the Fe-D vector being roughly along the Fe-N(O) direction. Because reduction of the Rieske cluster is required for O(2) binding and subsequent catalysis, the effect of its oxidation state on substrate binding was examined. The spectra from the NDO-naphthalene complex reveal two different binding conformations, which change in relative population when the oxidation state of the Rieske cluster is changed. This shift, and the conformational coupling it implies, may hold the key to both oxygen gating and oxygen reactivity for Rieske aromatic dioxygenases.

Dioxygenases↗

PubMed2003

Spectroscopic studies of the effect of ligand donor strength on the Fe-NO bond intradiol dioxygenases.

The geometric and electronic structure of NO bound to reduced protocatechuate 3,4-dioxygenase and its substrate (3,4-dihydroxybenzoate, PCA) complex have been examined by X-ray absorption (XAS), UV-vis absorption (Abs), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The results are compared to those previously published on model complexes described as [FeNO]7 systems in which an S = 5/2 ferric center is antiferromagnetically coupled to an S = 1 NO-. XAS pre-edge analysis indicates that the Fe-NO units in FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] lack the greatly increased pre-edge intensity representative of most [FeNO]7 model sites. Furthermore, from extended X-ray absorption fine structure (EXAFS) analysis, the FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] active sites are shown to have an Fe-NO distance of at least 1.91 A, approximately 0.2 A greater than those found in the model complexes. The weakened Fe-NO bond is consistent with the overall lengthening of the bond lengths and the fact that VTVH MCD data show that NO(-)-->FeIII CT transitions are no longer polarized along the z-axis of the zero-field splitting tensor. The weaker Fe-NO bond derives from the strong donor interaction of the endogenous phenolate and substrate catecholate ligands, which is observed from the increased intensity in the CT region relative to that of [FeNO]7 model complexes, and from the shift in XAS edge position to lower energy. As NO is an analogue of O2, the effect of endogenous ligand donor strength on the Fe-NO bond has important implications with respect to O2 activation by non-heme iron enzymes.

Algorithms↗

PubMed2002

Hydrogen peroxide-coupled cis-diol formation catalyzed by naphthalene 1,2-dioxygenase.

Naphthalene 1,2-dioxygenase (NDOS) catalyzes the NAD(P)H and O(2)-dependent oxidation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDOS consists of three protein components: a flavo-[2Fe-2S] reductase (NDR), a ferredoxin electron transfer protein (NDF), and an (alphabeta)(3) oxygenase (NDO) containing a mononuclear iron site and a Rieske-type [2Fe-2S] cluster in each alpha-subunit. The active site is built across a subunit-subunit boundary, and each subunit contributes one type of metal center. Our previous studies have shown that NDO with both metal centers reduced is capable of an O(2)-coupled single turnover to yield the correct cis-diol product in the absence of the NDR and NDF components (Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953). It is shown here that addition of H(2)O(2) to NDO allows reaction with naphthalene to rapidly yield the correct product in a "peroxide shunt" reaction that does not require a reduced Rieske cluster. The mononuclear Fe(2+) center is oxidized during turnover, while the Rieske cluster remains in the oxidized state. Peroxide shunt turnover in the presence of (18)O-labeled H(2)O(2), H(2)O, or O(2) shows that both oxygen atoms in the product derive primarily from H(2)O(2). The peroxide shunt halts after one turnover despite the presence of excess H(2)O(2) and naphthalene, but this is not the result of enzyme inactivation. Rather, it appears that the product cannot be released when the mononuclear iron is in the Fe(3+) state, blocking a second turnover. This work supports the hypotheses that the cis-dihydroxylation activity of NDOS requires only the NDO component, that a peroxo intermediate is formed during normal catalysis, and that product release requires an additional reducing equivalent beyond those necessary for the first turnover.

Catalysis↗

PubMed2002

Benzoate 1,2-dioxygenase from Pseudomonas putida: single turnover kinetics and regulation of a two-component Rieske dioxygenase.

The benzoate 1,2-dioxygenase system (BZDOS) from Pseudomonas putida mt-2 catalyzes the NADH-dependent oxidation of benzoate to 1-carboxy-1,2-cis-dihydroxycyclohexa-3,5-diene. Both the oxygenase (BZDO) and reductase (BZDR) components of BZDOS have been purified and characterized kinetically and by optical, EPR, and Mössbauer spectroscopies. BZDO has an (alpha beta)(3) subunit structure in which each alpha subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron site. Two different purification protocols were developed for BZDO allowing the mononuclear iron to be stabilized in either the Fe(III) or the Fe(II) state for spectroscopic characterization. Using single turnover reactions, it is shown that fully reduced BZDO alone is capable of yielding the cis-diol product in high yield at rates that exceed the BZDOS turnover number. At the conclusion of turnover, quantification of each oxidation state of the metal sites by EPR and Mössbauer spectroscopies shows that the Rieske cluster and mononuclear iron are each oxidized in amounts equal to the product yield, suggesting that the two electrons required for catalysis derive from the two metal centers. These results are in agreement with our previous study of naphthalene 1,2-dioxygenase [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953], which belongs to a different Rieske dioxygenase subclass, suggesting that it is a universal characteristic of Rieske dioxygenases that oxygen activation and substrate oxidation are catalyzed by the oxygenase component alone. The EPR spectrum of the Fe(III) center after a single turnover is distinct from either of those of substrate-free or substrate-bound enzyme. The complex with this spectrum is not formed by addition of cis-diol product to the resting Fe(III) form of the enzyme but is observed when the Fe(II) form is oxidized in the presence of product. Together, these results suggest that product exchange occurs only when the mononuclear iron is reduced. Stopped-flow and rapid scan analyses monitoring the oxidation of the Rieske cluster during the single turnover reaction show that it occurs in three phases that are kinetically competent for catalysis. The rate of each phase was found to be dependent on the type of substrate present, suggesting that the substrate influences the rate of electron transfer between the metal clusters. The participation of substrate in the oxygen activation reaction suggests a new aspect of the mechanism of this process by the Rieske dioxygenase class.

Electron Spin Resonance Spectroscopy↗

PubMed2002

Spectroscopic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: molecular mechanism and CO(2) activation in the biosynthesis of ethylene.

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyzes the last step in the biosynthesis of the gaseous plant hormone ethylene, which is involved in development, including germination, fruit ripening, and senescence. ACCO is a mononuclear non-heme ferrous enzyme that couples the oxidation of the cosubstrate ascorbate to the oxidation of substrate ACC by dioxygen. In addition to substrate and cosubstrate, ACCO requires the activator CO(2) for continuous turnover. NIR circular dichroism and magnetic circular dichroism spectroscopies have been used to probe the geometric and electronic structure of the ferrous active site in ACCO to obtain molecular-level insight into its catalytic mechanism. Resting ACCO/Fe(II) is coordinatively saturated (six-coordinate). In the presence of CO(2), one ferrous ligand is displaced to yield a five-coordinate site only when both the substrate ACC and cosubstrate ascorbate are bound to the enzyme. The open coordination position allows rapid O(2) activation for the oxidation of both substrates. In the absence of CO(2), ACC binding alone converts the site to five-coordinate, which would react with O(2) in the absence of ascorbate and quickly deactivate the enzyme. These studies show that ACCO employs a general strategy similar to other non-heme iron enzymes in terms of opening iron coordination sites at the appropriate time in the reaction cycle and define the role of CO(2) as stabilizing the six-coordinate ACCO/Fe(II)/ACC complex, thus preventing the uncoupled reaction that inactivates the enzyme.

Amino Acid Oxidoreductases↗

PubMed2002

Spectroscopic and electronic structure studies of protocatechuate 3,4-dioxygenase: nature of tyrosinate-Fe(III) bonds and their contribution to reactivity.

The geometric and electronic structure of the high-spin ferric active site of protocatechuate 3,4-dioxygenase (3,4-PCD) has been examined by absorption (Abs), circular dichroism (CD), magnetic CD (MCD), and variable-temperature-variable-field (VTVH) MCD spectroscopies. Density functional (DFT) and INDO/S-CI molecular orbital calculations provide complementary insight into the electronic structure of 3,4-PCD and allow an experimentally calibrated bonding scheme to be developed. Abs, CD, and MCD indicate that there are at least seven transitions below 35 000 cm(-1) which arise from tyrosinate ligand-to-metal-charge transfer (LMCT) transitions. VTVH MCD spectroscopy gives the polarizations of these LMCT bands in the principal axis system of the D-tensor, which is oriented relative to the molecular structure from the INDO/S-CI calculations. Three transitions are associated with the equatorial tyrosinate and four with the axial tyrosinate. This large number of transitions per tyrosinate is due to the pi and importantly the sigma overlap of the two tyrosinate valence orbitals with the metal d orbitals and is governed by the Fe-O-C angle and the Fe-O-C-C dihedral angles. The previously reported crystal structure indicates that the Fe-O-C angles are 133 degrees and 148 degrees for the equatorial and axial tyrosinate, respectively. Each tyrosinate has transitions at different energies with different intensities, which correlate with differences in geometry that reflect pseudo-sigma bonding to the Fe(III) and relate to reactivity. These factors reflect the metal-ligand bond strength and indicate that the axial tyrosinate-Fe(III) bond is weaker than the equatorial tyrosinate-Fe(III) bond. Furthermore, it is found that the differences in geometry, and hence electronic structure, are imposed by the protein. The consequences to catalysis are significant because the axial tyrosinate has been shown to dissociate upon substrate binding and the equatorial tyrosinate in the enzyme-substrate complex is thought to influence asymmetric binding of the chelated substrate moiety via a strong trans influence which activates the substrate for reaction with O2.

Binding Sites↗