Immobilized metal complexes in porous organic hosts: development of a material for the selective and reversible binding of nitric oxide.

Delivery of NO to specific targets is important in fundamental studies and therapeutic applications. Various methods have been reported for delivery of NO in vivo and in vitro; however, there are few examples of systems that reversibly bind NO. Reported herein is the development of a new polymer (P-1[Co(II)]) that reversibly binds NO. P-1[Co(II)] has a significantly higher affinity for NO compared to O(2), CO(2), and CO. The polymer is synthesized by template copolymerization methods and consists of a porous methacrylate network, containing immobilized four-coordinate Co(II) sites. Binding of NO causes an immediate color change, indicating coordination of NO to the site-isolated Co(II) centers. The formation of P-1[Co(NO)] has been confirmed by EPR, electronic absorbance, and X-ray absorption spectroscopies. Electronic and X-ray absorbance results for P-1[Co(II)] and P-1[Co(NO)] show that the coordination geometry of the immobilized cobalt complexes are similar to those of their monomeric analogues and that NO binds directly to the cobalt centers. EPR spectra show that the binding of NO to P-1[Co(II)] is reversible in the solid state; the axial EPR signal associated with the four-coordinate Co(II) sites in P-1[Co(II)] is quenched upon NO binding. At room temperature and atmospheric pressure, 40% conversion of P-1[Co(NO)] to P-1[Co(II)] is achieved in 14 days; under vacuum at 120 degrees C this conversion is complete in approximately 1 h. The binding of NO to P-1[Co(II)] is also observed when the polymer is suspended in liquids, including water.

Porphobilinogen synthase from pea: expression from an artificial gene, kinetic characterization, and novel implications for subunit interactions.

Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin B(12). The homooctameric metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen. An artificial gene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic oligonucleotides. The recombinant 330 residue enzyme without a putative chloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities. The specific activity is protein concentration dependent, which indicates that a maximally active octamer can dissociate into less active smaller units. The enzyme is most active at slightly alkaline pH; it shows two pK(a) values of 7.4 and 9.7. Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit; kinetic data support two functionally distinct types of Mg(II) and the third appears to be nonphysiologic and inhibitory. Analysis of the protein concentration dependence of the specific activity suggests that the minimal functional unit is a tetramer. A model of octameric pea PBGS was built to predict the location of intermolecular disulfide linkages that were revealed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As verified by site-specific mutagenesis, disulfide linkages can form between four cysteines per octamer, each located five amino acids from the C-terminus. These data are consistent with the protein undergoing conformational changes and the idea that whole-body motion can occur between subunits.

Protonation of porphyrin in iron-free cytochrome c: spectral properties of monocation free base porphyrin, a charge analogue of ferric heme.

Charged groups reside mainly on protein surfaces, but for proteins that incorporate redox centers, a charge typically exists at the prosthetic group within the interior. How a protein accommodates a buried charge and the effect of redox changes on protein stability are thermodynamically related problems. To examine these problems in cytochrome c, the metal-free protein was used as a model. When pH is lowered, the neutral, monocation, and dication forms of the porphyrin are progressively formed as indicated by their characteristic absorption spectra. Infrared studies of the protein over this pH range show that the protein remains in a predominately alpha-helical structure, although the carboxyl groups of the dicarboxylic amino acids become protonated at lower pH. The monocation porphyrin form (which has not been previously reported in a protein and is a charge analogue of ferric heme) has a fluorescence maximum at 609 nm. The pKs for the respective one and two protonation of the porphyrin pyrrole Ns are 3.2 and 1.6 for the folded protein, and 4.4 and 3.1 for the unfolded protein. These values indicate that the protection of the polypeptide chain for protonation is approximately 3 kcal.

X-ray spectroscopy of nitrile hydratase at pH 7 and 9.

The iron K-edge X-ray absorption spectrum of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase in frozen solutions at pH 7 and 9 has been analyzed to determine details of the iron coordination. EXAFS analysis implies two or three sulfur ligands per iron and overall six coordination; together with previous EPR and ENDOR results, this implies an N3S2O ligation sphere. The bond lengths from EXAFS analysis [rav(Fe-S) = 2.21 A at pH 7.3; rav(Fe-N/O) = 1.99 A] support cis coordination of two cysteine ligands and conclusively rule out nitric oxide coordination to the iron, a possibility proposed on the basis of an FTIR difference experiment [Noguchi, T., Honda, J., Nagamune, T., Sasabe, H., Inoue, Y., & Endo, I. (1995) FEBS Lett. 358, 9-12]. The higher-frequency EXAFS can be simulated well by inclusion of multiple scattering from two or three imidazole ligands; the fit to the data is improved if first-sphere multiple scattering pathways are also included. A slight shortening (by 0.02 +/- 0.01 A) of one or both Fe-S bonds when the pH is raised from 7.3 to 9.0 is consistent with shifts observed in the Raman spectrum [Brennan et al. (1996) Biochemistry 35, 10068-10077].

Circular dichroism and x-ray spectroscopies of Azotobacter vinelandii nitrogenase iron protein. MgATP and MgADP induced protein conformational changes affecting the [4Fe-4S] cluster and characterization of a [2Fe-2S] form.

Nucleotide interactions with nitrogenase are a central part of the mechanism of nitrogen reduction. Previous studies have suggested that MgATP or MgADP binding to the nitrogenase iron protein (Fe protein) induce protein conformational changes that control component protein docking, interprotein electron transfer, and substrate reduction. In the present study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray absorption spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising from transitions of the [4Fe-4S]2+ cluster. We present evidence that MgADP or MgATP binding to the oxidized nitrogenase Fe protein results in distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fc-4S] cluster. The present results are consistent with previous studies such as chelation assays, electron paramagnetic resonance, and NMR, which suggested that MgADP or MgATP binding to the nitrogenase Fe protein induced different conformational changes. The CD spectrum of a [2Fe-2S]2+ form of the nitrogenase Fe protein was also investigated to address the possibility that the MgATP- or MgADP-induced changes in the CD spectrum of the native enzyme were the result of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence was found for a contribution of a [2Fe-2S]2+ cluster to the CD spectrum of oxidized Fe protein in the absence or presence of nucleotides. A novel two-electron reduction of the [2Fe-2S]2+ cluster in Fe protein was apparent from absorption, CD, and electron paramagnetic resonance data. Fe K-edge x-ray absorption spectra of the oxidized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein. The present results reveal that MgATP or MgADP binding to the oxidized state of the Fe protein result in different conformational changes in the environment around the [4Fe-4S] cluster.

Structure and kinetics of formation of catechol complexes of ferric soybean lipoxygenase-1.

Ferric soybean lipoxygenase forms stable complexes with 4-substituted catechols. The structure of the complex between the enzyme and 3,4-dihydroxybenzonitrile has been studied by resonance Raman, electron paramagnetic resonance, visible, and X-ray spectroscopies. It is a bidentate iron-catecholate complex with at least one water ligand. The kinetics of formation of complexes between lipoxygenase and 3,4-dihydroxybenzonitrile and 3,4-dihydroxyacetophenone have been studied by stopped-flow spectroscopy. The data are consistent with two kinetically distinct, reversible steps. The pH dependence of the first step suggests that the substrate for the reaction is the catechol monoanion. When these results are combined, plausible mechanisms for the complexation reaction are suggested.

X-ray spectroscopy of the iron site in soybean lipoxygenase-1: changes in coordination upon oxidation or addition of methanol.

Iron K-edge X-ray spectroscopy (XANES and EXAFS) was used to study iron coordination in frozen solutions of soybean lipoxygenase-1 (SLO). The intensity of the 1s-->3d pre-edge transition of native iron(II) lipoxygenase is greater than what was found for six-coordinate high-spin iron(II) model complexes, but comparable to that of a five-coordinate model. This and a relatively short average bond length determined by EXAFS (2.13 A) indicate that the native lipoxygenase in our frozen samples is five-coordinate, excluding possible bonds longer than 2.5 A. The coordination of the iron(II) in native lipoxygenase changes when methanol (as low as 0.1%) or glycerol (20%) is added to the buffer prior to freezing. The addition of methanol diminishes the pre-edge transition and increases EXAFS-derived bond lengths by 0.04 A, indicating a change to six-coordination. The small pre-edge feature in active iron(III) lipoxygenase suggests six-coordination. EXAFS indicates a short, 1.88 A Fe-O bond, which, given other spectroscopic and crystallographic evidence, is assigned to coordinated hydroxide. The average of the remaining bond lengths is 2.11 A. The iron coordination in iron(III) lipoxygenase is less affected by the presence of alcohols than is the site in the iron(II) enzyme. Bond valence sums indicate that the bond lengths for lipoxygenase derived from our EXAFS analyses are comparable to those of crystallographically characterized model complexes. The flexibility of the coordination number in SLON (native SLO) and the presence of an [FeIIIOH]2+ unit in SLOA (active SLO) are of possible mechanistic importance.