VTVH MCD Research Articles

Nature of the intermediate formed in the reduction of O(2) to H(2)O at the trinuclear copper cluster active site in native laccase.

The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.

Spectroscopic studies of substrate interactions with clavaminate synthase 2, a multifunctional alpha-KG-dependent non-heme iron enzyme: correlation with mechanisms and reactivities.

Using a single ferrous active site, clavaminate synthase 2 (CS2) activates O(2) and catalyzes the hydroxylation of deoxyguanidinoproclavaminic acid (DGPC), the oxidative ring closure of proclavaminic acid (PC), and the desaturation of dihydroclavaminic acid (and a substrate analogue, deoxyproclavaminic acid (DPC)), each coupled to the oxidative decarboxylation of cosubstrate, alpha-ketoglutarate (alpha-KG). CS2 can also catalyze an uncoupled decarboxylation of alpha-KG both in the absence and in the presence of substrate, which results in enzyme deactivation. Resting CS2/Fe(II) has a six-coordinate Fe(II) site, and alpha-KG binds to the iron in a bidentate mode. The active site becomes five-coordinate only when both substrate and alpha-KG are bound, the latter still in a bidentate mode. Absorption, CD, MCD, and VTVH MCD studies of the interaction of CS2 with DGPC, PC, and DPC provide significant molecular level insight into the structure/function correlations of this multifunctional enzyme. There are varying amounts of six-coordinate ferrous species in the substrate complexes, which correlate to the uncoupled reaction. Five-coordinate ferrous species with similar geometric and electronic structures are present for all three substrate/alpha-KG complexes. Coordinative unsaturation of the Fe(II) in the presence of both cosubstrate and substrate appears to be critical for the coupling of the oxidative decarboxylation of alpha-KG to the different substrate oxidation reactions. In addition to the substrate orientation relative to the open coordination position on the iron site, it is hypothesized that the enzyme can affect the nature of the reactivity by further regulating the binding energy of the water to the ferrous species in the enzyme/succinate/product complex.

Near-infrared circular dichroism and magnetic circular dichroism and x-ray absorption spectral comparison of the non-heme ferrous active sites of plant and mammalian 15-lipoxygenases

Lipoxygenases (LOs) are non-heme iron enzymes which catalyze the reaction of dioxygen with cis,cis- 1,4-pentadiene-containing fatty acids to form hydroperoxide products, which in mammals are the precursors to the inflammation- and immunity-mediating compounds lipoxins and leukotrienes. Recent X-ray crystal structures of ferrous soybean lipoxygenase-1 (SLO-I) offer two different descriptions of the active site: one four-coordinate and one five- or six-coordinate. Near-infrared (NfIR) circular and magnetic circular dichroism (CD/MCD) and variable- temperature, variable-field (VTVH) MCD have been used to study SLO-I in solution which is found to exist as a 40/60% mixture of five- and six-coordinate forms, respectively. Addition of linoleate substrate or alcohols shifts the mixture to the purely six-coordinate form. NIR CD/MCD studies of two mammalian LOs, rabbit reticulocyte and recombinant human 15-LOs, show that these exist as pure six-coordinate forms. X-ray absorption Fe K-edge and pre-edge data also show that the mammalian 15-LOs and SLO-I in glycerol are six-coordinate. This is consistent with the extended X-ray absorption fine structure (EXAFS) results of SLO-I in glycerol which show the iron active site to have 5 ± 1 N/0 at ~2.16 A. VTVH MCD data on the six-coordinate sites show that the mammalian and soybean enzymes have very different ground-state splittings, indicative of differences in bonding interactions with the ligand set. These differences in ferrous site coordination in solution and ground-state splittings are attributed to the substitution of a stronger histidine ligand in the mammalian 15-LOs for an asparagine in SLO-I.