Substrate binding to NO-ferro-naphthalene 1,2-dioxygenase studied by high-resolution Q-band pulsed 2H-ENDOR spectroscopy

The active site 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 (electron nuclear double resonance) spectra have been recorded for the NO-ferrous center of NDO bound with the substrates d(8)-naphthalene, d(2)-naphthalene, d(8)-toluene, d(3)-toluene, and d(6)-benzene; samples were prepared in a D(2)O buffer to test for solvent-derived ligands; spectra were collected for enzymes with the Rieske diiron center in both its oxidized and reduced states. A sharp quartet ENDOR pattern from a nearby deuteron of the substrate in a major binding geometry (denoted as A) was detected for all perdeuterated substrates. Examination of the sample prepared with 1,4-di-deutero-naphthalene shows that the signal arises from D1. Analysis of two-dimensional (2-D) orientation-selective ENDOR patterns collected for this sample defined the location of the D1 deuteron, with respect to the g-frame of the iron center and the orientation of the C-D1 bond. Consideration of the orientations of naphthalene that are permitted within the constraints of these results, as supported by a novel approach to simulations of orientation-selective, 2-D ENDOR patterns for the perdeuterated naphthalene sample, which summed contributions from D1/D2/D8, disclose the geometry of the naphthalene and the Fe-NO fragment. The two deuterons of the reactive carbons, D1 and D2, are closest to the Fe atom (r(Fe)(-)(D1) approximately 4.3 A, r(Fe)(-)(D2) approximately 5.0 A), whereas D8 is farther away (r(Fe)(-)(D8) approximately 5.3 A). Perhaps more instructive, D1-N and D2-N distances to the O(2) surrogate, NO, are approximately 2.4 and approximately 3.3 A, respectively, whereas the D8-N distance is approximately 3.7 A. The data show that benzene and the aromatic ring of toluene also sit within the substrate-binding pocket adjacent to the mononuclear Fe atom. These rings occupy a position similar to that of the "proximal" ring of naphthalene, with the closest ring deuteron being located at a distance of approximately 4.3-4.4 A from the Fe atom and with the Fe-D vector being slightly off the Fe-N(O) direction. In particular, comparison of the data for d(8)-toluene and methyl-d(3)-toluene shows that the methyl group of toluene points away from the Fe atom, despite observations that the oxidation of toluene occurs at the methyl group during catalysis. The Rieske cluster is reduced during both steady-state and single-turnover catalysis; therefore, the effect of its oxidation state on the geometry of substrate binding was examined. The spectra from the NDO-naphthalene complex also revealed a second binding conformation (denoted as B), in which the substrate is located approximately 0.5 A farther from the Fe atom. The relative populations of A- and B-sites are allosterically changed when the Rieske cluster is reduced. ENDOR of exchangeable protons shows that the water/hydroxide of Fe-NDO is retained upon binding NO.     

Spectrophotometric determination of anionic surfactants by conversion of the leuco-bases of triphenylmethane dyes

A one-phase, one-reagent, spectrophotometric technique is reported for the detection and determination of anionic surfactants of the sulfonate and sulfate types. Both applications are based on conversion of the colorless leuco-bases of triphenylmethane dyes (Brilliant Green and methyl violet) to colored (quinoid) forms by the anionic surfactants. The quantitative method, in the absorption and reflectance modes, is applicable to anionic surfactants in the 0–1500 ppm range; the spectrophotometric procedure can be automated. Residual surfactant can be detected at the 0.01% level on the surface of cellulosic products.     

Synthesis and reactions of aminoporphyrazines with annulated five- and seven-membered rings

The novel five- and seven-membered ring appended aminoporphyrazines 3 and 12 have been prepared via mixed Linstead macrocyclization. The structures of both have been unequivocally established by X-ray crystallographic studies. Reductive deselenation of selenadiazole 3 in the presence of 9,10-phenanthrenequinone or 2,3-butanedione results in the formation of pyrazines 6a,b, whereas oxidation of porphyrazine 12 gave the corresponding seco derivative 14. seco-Porphyrazine 14 mediates the generation of singlet oxygen with a quantum yield of 0.74.     

Proton transfer at helium temperatures during dioxygen activation by heme monooxygenases

In the first measurement of enzymatic proton transfer at liquid helium temperatures, we examine protonation of the peroxo-ferriheme state of heme oxygenase (HO) produced by in situ radiolytic cryoreduction of oxy-HO in H2O and D2O solvents at ca. 4 K and above, and compare these findings with analogous measurements for oxy-P450cam and for oxy-Mb. Proton transfer in HO occurs at helium temperatures in both solvents; it occurs in P450cam at approximately 50 K and higher; in Mb it does not occur until T > 170 K. For Mb, this transfer at 180 K is biphasic, and the majority phase shows a solvent kinetic isotope effect of 3.8. We discuss these results in the context of the picture of environmentally coupled tunneling, which links proton transfer to two classes of protein motions: environmental reorganization (lambda in Marcus-like equations) and protein fluctuations ("active dynamics"; gating) which modulate the distance of proton transfer.     

A novel immunoassay system and bioseparation process based on thermal phase separating polymers

Poly-N-isopropylacrylamide (polyNIPAAm), a water-soluble, thermally precipitating synthetic polymer, has been conjugated together with a monoclonal antibody (MAb) and utilized in a novel separation method for an immunoassay. The PolyMPAAm precipitates out of water above a critical temperature of 31°C, enabling a polymerbound immune complex to be separated from the solution. The principal advantages of this method are that it utilizes a homogeneous incubation for the antigen-antibody reaction, plus, it has the ability to assay large-molecular-weight antigens with sensitivities equivalent to other nonisotopic heterogeneous immunoassays. In addition, since the polymer-immune complex may be reversibly redissolved by cooling, the method may be used both to concentrate the signal and isolate the analyte. This general technique may also be used for a wide variety of separation processes in addition to immunoassays, in which a specific component in a biological fluid, industrial process stream, or body of water is to be isolated for analysis, recovery, or disposal. Thus, product recovery and/or toxin or pollutant removal processes are possible with this methodology.     

Large-scale 0–1 linear programming on distributed workstations

We present a methodology which uses a collection of workstations connected by an Ethernet network as a parallel processor for solving large-scale linear programming problems. On the largest problems we tested, linear and super-linear speedups have been achieved. Using the branch-and-cut approach of Hoffman, Padberg and Rinaldi, eight workstations connected in parallel solve problems from the test set documented in the Crowder, Johnson and Padberg 1983Operations Research article. Very inexpensive, networked workstations are now solving in minutes problems which were once considered not solvable in economically feasible times. In this peer-to-peer (as opposed to master-worker) implementation, interprocess communication was accomplished by using shared files and resource locks. Effective communication between processes was accomplished with a minimum of overhead (never more than 8% of total processing time). The implementation procedures and computational results will be presented.Supported in part by a grant from the Digital Equipment Corporation.Supported in part by grants from the Office of Naval Research and the National Science Foundation (ECS-8615438).     

Determination of the entropy-factor of the gold donor level in silicon by resistivity and DLTS measurements

The parameters of the gold donor level inp-type silicon are determined from the temperature dependence of the free carrier concentration and from DLTS measurements. The entropy-factorX _T is determined to be 20±2. In addition, the capture cross-section for holes _p =5.5×10^–15 cm² and the reaction enthalpyH _T –H _v =0.35eV for the exchange of holes between the gold donor level and the valence band are obtained in the present investigation.     

A superoxo-ferrous state in a reduced oxy-ferrous hemoprotein and model compounds

Cryoreduction of the [FeO2]6 (n = 6 is the number of electrons in 3d orbitals on Fe and pi* orbitals on O2) dioxygen-bound ferroheme through irradiation at 77 K generates an [FeO2]7 reduced oxy-heme. Numerous investigations have examined [FeO2]7 centers that have been characterized as peroxo-ferric centers, denoted [FeO2]per7, in which a ferriheme binds a dianionic peroxo-ligand. The generation of such an intermediate can be understood heuristically if the [FeO2]6 parent is viewed as a superoxo-ferric center and the injected electron localizes on the O-O moiety. We here report EPR/ENDOR experiments which show quite different properties for the [FeO2]7 centers produced by cryoreduction of monomeric oxy-hemoglobin (oxy-GMH3) from Glycera dibranchiata, which is unlike mammalian "globins" in having a leucine in place of the distal histidine; of frozen aprotic solutions of oxy-ferrous octaethyl porphyrin; and of the oxy-ferrous complex of the heme model, cyclidene. These [FeO2]7 centers are characterized as "superoxo-ferrous" centers ([FeO2]sup7), with nearly unit spin density localized on a superoxo moiety which is end-on coordinated to a low-spin ferrous ion. This assignment is based on their g tensors and 17O hyperfine couplings, which are characteristic of the superoxide ion coordinated to a diamagnetic metal ion, and on the absence of detectable ENDOR signals either from the in-plane 14N ligands or from an exchangeable H-bond proton. Such a center would arise if the electron that adds to the [FeO2]6 superoxo-ferric parent localizes on the Fe ion, to make a superoxo-ferrous moiety. Upon annealing to T > 150 K, the [FeO2]sup7 species converts to peroxo/hydroperoxo-ferric ([FeO2H]7) intermediates. These experiments suggest that the primary reduction product is [FeO2]sup7 and that the internal redox transition to [FeO2]per7/[FeO2H]7 states is driven at least in part by H-bonding/proton donation by the environment.