Robert C. Scarrow

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] 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 [4Fe-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] 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] 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] 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.

How does cyanide inhibit superoxide reductase?Insight from synthetic FeIIIN4S model complexes

Superoxide reductases (SORs) are nonheme iron-containing enzymes that reduce HO2 to H2O2. Exogenous substrates such as N\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{3}^{-}\end{equation*}\end{document} and CN− have been shown to bind to the catalytic iron site of SOR, and cyanide acts as an inhibitor. To understand how these exogenous ligands alter the physical and reactivity properties of the SOR iron site, acetate-, azide-, and cyanide-ligated synthetic models of SOR have been prepared. The x-ray crystal structures of azide-ligated [FeIII(SMe2N4(tren))(N3)]+ (3), dimeric cyanide-bridged ([FeIII(SMe2N4(tren))]2-μ-CN)3+ (5), and acetate-ligated [FeIII(SMe2N4(tren))(OAc)]+ (6) are described, in addition to x-ray absorption spectrum-derived and preliminary crystallographic structures of cyanide-ligated [FeIII(SMe2N4(tren))(CN)]+ (4). Cyanide coordination to our model (4) causes the redox potential to shift anodically by 470 mV relative to acetate-ligated 6 and 395 mV relative to azide-ligated 3. If cyanide coordination were to cause a similar shift in redox potential with SOR, then the reduction potential of the catalytically active Fe3+ center would fall well below that of its biological reductants. These results suggest therefore that cyanide inhibits SOR activity by making the Fe2+ state inaccessible and thus preventing the enzyme from turning over. Cyanide inhibits activity in the metalloenzyme superoxide dismutase via a similar mechanism. The reduced five-coordinate precursor to 3, 4, and 6 [FeII(SMe2N4(tren))]+ (1) was previously shown by us to react with superoxide to afford H2O2 via an [FeIII(SMe2N4(tren))(OOH)]+ intermediate. Cyanide and azide do not bind to 1 and do not prevent 1 from reducing superoxide.

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.