Vinosum High-potential Iron Protein Research Articles

Elucidation of a [4Fe-4S] cluster degradation pathway: rapid kinetic studies of the degradation of Chromatium vinosum HiPIP.

Irreversible disassembly of the 4Fe-4S cluster in Chromatium vinosum high-potential iron protein (HiPIP) has been investigated in the presence of a low concentration of guanidinium hydrochloride. From the dependence of degradation rate on [H+], it is deduced that at least three protons are required to trigger efficient cluster degradation. Under these conditions the protonated cluster shows broadened Mössbauer signals, but delta EQ (1.1 mm/s) and delta (0.44 mm/s) are similar to the native form. Collapse of the protonated transition state complex, revealed by rapid-quench Mössbauer experiments, occurs with a measured rate constant kobs approximately 0.72 +/- 0.35 s-1 that is consistent with results from time-resolved electronic absorption and fluorescence (kobs approximately 0.4 +/- 0.1 s-1) and EPR (kobs approximately 0.62 +/- 0.18 s-1) measurements. Apparently, guanidinium hydrochloride serves to perturb the tertiary structure of the protein, facilitating protonation of the cluster, but not degradation per se. Release of iron ions occurs even more slowly with kobs approximately 0.07 +/- 0.02 s-1, as determined by the appearance of the g = 4.3 EPR signal. Proton-mediated cluster degradation is sensitive to the oxidation state of the cluster, with the oxidized state showing a two-fold slower rate in acidic solutions as a result of increased electrostatic repulsion with the cluster. Consistent results are obtained from absorption, fluorescence, Mössbauer and EPR measurements.

Functional role of a conserved tryptophan residue of Chromatium vinosum high potential iron protein

A Trp80Asn point mutation of Chromatium vinosum high potential iron protein has provided insight on the functional role of this conserved aromatic residue. Following mutation to Asn, a minimal change in the midpoint potential of the [Fe4S4]3+/2+ cluster is observed (+33 mV). In contrast to other proteins carrying mutations of Tyr and Phe residues, the reduced state of the Trp80Asn derivative is stable in an aerobic environment, but displays a more pronounced temperature-dependent degradation that appears to be mediated by unfolding of the protein prior to degradation of the cluster. Accordingly, Trp80 serves a distinct role from other aromatic residues found in the hydrophobic cluster binding pocket. The rate of electron transfer exchange with ferricyanide is similar to that observed for native protein (knat=0.7 s−1; kTrp80Asn=1.7 s−1), the minor increase in reactivity reflecting the change in driving force from the increase in cluster midpoint potential.

Chemistry of Nitric Oxide with Protein-Bound Iron Sulfur Centers. Insights on Physiological Reactivity

The anaerobic reaction of C. vinosum high-potential iron protein (HiPIP) with nitric oxide has been studied in order to understand the chemical reactivity of NO with protein-bound iron−sulfur clusters. Despite having a solvent inaccessible 4Fe-4S center, native HiPIP reacts with diethylamineNONOate (an NO donor), resulting in protein unfolding and the formation of protein-bound dinitrosyl−iron complexes (DNICs) with a typical gav = 2.03 EPR signal. These cysteinyl-coordinated DNICs are directly observed for the first time by use of electrospray ionization−mass spectrometry (ESI-MS) and are found only in a stoichiometry of 2:1 DNIC:protein. Our results suggest that these complexes form only when the protein is folded. ESI-MS also demonstrates that NO-mediated cluster degradation results in nitrosation of the protein at sites other than cysteine. Finally, reactivity comparison of native and Tyr19Leu HiPIP demonstrates solvent accessibility to be an important, but not necessary factor for Fe−S cluster degrad...

Phenylalanine 48 of Chromatium vinosum high potential iron protein is essential for stability of the oxidized [Fe 4S 4] cluster. Site-directed mutagenesis and NMR studies as a probe of cluster chemistry

Point mutations of the conserved aromatic residue phenylalanide 48 (Phe48Arg.His) in Chromatium vinosum high potential iron protein have been examined with the aim of understanding the functional role of this residue. For both mutants the change in midpoint potential of the [Fe 4S 4] 3+/2+ cluster is minimal (< + 20 mV). The oxidized state is unstable relative to the recombinant native, with the observation of an ‘autoreduction’ pathway that appears to be mediated by degradation of the cluster in a fraction of the oxidized sample. This provides the reducing equivalents required to bring about reduction of the remainder of the sample. This degradative reaction results from the increased solvent accessibility of the cluster core in the non-conservative Phe48 mutants. Increased solvent access has been characterized by 1H 15N HMQC experiments.

Factors Influencing Redox Thermodynamics and Electron Self-Exchange for the [Fe4S4] Cluster in Chromatium vinosum High Potential Iron Protein: The Role of Core Aromatic Residues in Defining Cluster Redox Chemistry

The roles of aromatic core residues in regulating the reduction potential, the enthalpy and entropy of reduction, and the self-exchange rate constants for electron-transfer reactions for the prosthetic [Fe4S4]3+/2+ cluster of Chromatium vinosum high potential iron protein (HiPIP) have been addressed by a combination of site-directed mutagenesis, high field NMR (EXSY) experiments, and variable temperature spectrochemical redox titration measurements. Minimal changes are observed following nonconservative mutation of residues Tyr19, Phe48, and Phe66. Apparently these hydrophobic residues play only a minor role in defining the electronic properties of the cluster. These data support a model, first defined from results obtained on Tyr19 mutant HiPIP's [Agarwal, A., Li, D., & Cowan, J.A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9440-9444], in which the aromatic core restricts solvent accessibility and thereby stabilizes the oxidized [Fe4S4]3+ cluster.

Evaluation of Solvent Accessibility to the [Fe4S4] Binding Pocket in Native and Tyr19 Mutant High Potential Iron Proteins by 1H−15N HMQC and 19F NMR Experiments

The solvent accessibility of Chromatium vinosumhigh potential iron protein (HiPIP) has been investigated by use of (1)H-(15)N HMQC, and (19)F NMR spectroscopy. These NMR experiments indicate that solvent accessibility to the cluster core is similar, and minimal, for the reduced and oxidized states of native HiPIP, but increases significantly for mutant proteins (Tyr19Leu and Tyr19His). These results support a proposed role [Agarwal, A.; Li, D.; Cowan, J. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9440-9444] for Tyr19 in maintaining hydrolytic stability of the [Fe(4)S(4)] cluster, and demonstrate a general strategy for mapping out the solvent accessibility of protein-bound metalloredox prosthetic centers.

19F NMR Studies of Fluorine-Labeled Chromatium vinosum High-Potential Iron Protein

Fluorine-labeled analogues of Chromatium vinosum high-potential iron protein have been investigated by 19F NMR spectroscopy. Fluorine-19 resonances have been assigned, and chemical shift variations, relaxation times, and temperature dependencies have been determined. Observed changes in fluorine chemical shifts and relaxation times following oxidation of the [Fe4S4] cofactor appear to reflect structural perturbation of the protein backbone and side chains, rather than variations in the paramagnetism of the cluster. Fluorine-19 NMR provides a probe of redox-dependent conformational change in electron-transfer proteins, which may also be of value for structural characterization of mutants. Evaluation of H2O/D2O solvent isotope effects on 19F chemical shifts reflects solvent accessibility to various protein domains, while measurement of 19F relaxation times affords a convenient test of the relative contribution of cross-relaxation to magnetization decay. For HiPIP, the results reported herein indicate that the cross-relaxation contribution to the longitudinal relaxation (T1) is relatively small for both the oxidized and reduced states. Unusual temperature dependencies and fast relaxation times for the 19F resonances of 3-F-Phe66 and 3-F-Tyr19 labeled HiPIP support a close interaction of these two residues with the iron−sulfur cluster.

15N resonance assignments of oxidized and reducedChromatium vinosum high-potential iron protein

The 15N resonances in reduced and oxidized Chromatium vinosum high-potential iron protein have been assigned by use of 1H-1H COSY spectra and 1H-15N HMQC. HMQC-COSY, and HMQC-NOESY spectra. Unambiguous assignment of 70 of 85 backbone 15N resonances in the reduced protein and 62 of 85 resonances in the oxidized protein are made, as are 12 of 21 side-chain 15N resonances.

Calculation of the Redox Potentials of Iron-Sulfur Proteins: The 2-/3- Couple of [Fe4S4Cys4] Clusters in Peptococcus aerogenes Ferredoxin, Azotobacter vinelandii Ferredoxin I, and Chromatium vinosum High-Potential Iron Protein

Calculations of the redox potentials of the 2-/3-couples of [Fe4S*4Cys4] clusters in the iron-sulfur proteins Peptococcus aerogenes ferredoxin (PaFd), Azotobacter vinelandii ferredoxin I (AvFdI) and Chromatium vinosum high potential iron protein (CvHiPIP) based on the Protein Dipoles Langevin Dipoles (PDLD) method are reported. The structures of these proteins have been determined by X-ray crystallography; in the case of PaFd the structure has recently been revised due to a change in the sequence close to Cluster II. The large differences between the potentials of the [Fe4S*4Cys4] clusters of PaFd and AvFdI and the potential of the [Fe4S*4Cys4] cluster of CvHiPIP are successfully modeled and originate principally in differences in the configuration of main-chain amide groups near the clusters. The small difference between the potentials of PaFd and AvFdI is also satisfactorily modeled in the case of Cluster I of PaFd. Solvent dipoles close to the cluster in PaFd are an important contributor to its higher potential. The two X-ray structures of PaFd yield similar results for Cluster I of PaFd. In contrast, the results for Cluster II differ substantially; for reasons not yet clear, the recently revised structure leads to results in worse agreement with experiment.

Studies of the Electronic and Dynamic Properties of High-Potential Iron Proteins by Substitution with Non-Natural Amino Acids. 3-Fluorotyrosine-Modified Chromatium vinosum High-Potential Iron Protein

Studies of the Electronic and Dynamic Properties of High-Potential Iron Proteins by Substitution with Non-Natural Amino Acids. 3-Fluorotyrosine-Modified Chromatium vinosum High-Potential Iron Protein

Electronic structure of the [Fe4Se4]3+ clusters in C. vinosum HiPIP and Ectothiorhodospiza halophila HiPIP II through NMR and EPR studies

The selenium-for-sulfur substitutions in the [4Fe-4S] core of the high-potential iron protein (HiPIP) from Chromatium vinosum and HiPIP II from Ectothiorhodospira halophila were carried out, and the modified proteins were characterized by electronic EPR and NMR spectroscopies. EPR spectra showed a small increase in anisotropy and average g values. These results were interpreted as an indication that the Se-for-S substitution does not influence significantly the properties of the spin ground state. The assignment of the hyperfine shifted signals corresponding to protons of the four cysteine residues bound to the iron ions of the [4Fe-4Se] core was performed for both proteins using difference NOE, NOESY, EXSY, and TOCSY 1 H NMR methods

Molecular modeling studies on the proposed NaCl-induced dimerization of Chromatium vinosum high-potential iron protein

Previous work (Dunham, W.R., Hagen, W.R., Fee, J. A., Sands, R.H., Dunbar, J.B., Humblet, C. (1991) An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mössbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions, Biochimica Biophysica Acta 1079, 253–262) suggested that under specific solution conditions and slow freezing times, samples of oxidized Chromatium vinosum ( C v ) high-potential, iron-sulfur protein (HiPIP) form dimeric structures that exhibit characteristic spin-spin interaction in the EPR spectrum. In that study, it was also shown that two HiPIP molecules could approach each other along their Fe1-S4 axes to a distance of ∼ 13–14 A ̊ , as required by an analysis of the spin-spin physics. This is made possible because of a flattened surface on one side of the molecule within which S4 may, depending on side-chain motions, interact with solvent (Carter, C.W., Jr., Kraut, J., Freer, S.T., Alden, R.A., Sieker, L.C., Adman, E.T., Jensen, L.H. (1972) A comparison of Fe 4S 4 clusters in high potential iron protein and in ferredoxin, Proc. Natl. Acad. Sci. USA 69, 3527-3529). Here we describe a computer generated, hypothetical model of this proposed dimeric structure which suggests an energetically favorable interaction between two C v HiPIP molecules and could account for the experimental observations. Two C v HiPIP molecules brought together along their Fe1-S4 axes and maintained at a center-to-center distance of 14 Å can be rotated with respect to each other so as to create complementary interactions between two glutamine residues, two phenylalanine residues, and two leucine residues, and an energetically unfavorable interaction between two arginine residues. Energy minimization calculations using the program XPLOR indicate that this arrangement may provide an overall energetically favorable interaction between the two HiPIP molecules that is strengthened by site-specific binding of Na and Cl ions.

Polarographic studies of Chromatium vinosum high-potential iron protein : Evidence for surface chemistry at the Hg-electrode

Polarographic studies of Chromatium vinosum high-potential iron protein : Evidence for surface chemistry at the Hg-electrode

Polarographic studies of Chromatium vinosum high-potential iron protein : Evidence for surface chemistry at the Hg-electrode

Polarographic studies of Chromatium vinosum high-potential iron protein : Evidence for surface chemistry at the Hg-electrode

Proton NMR spectra and electron-transfer properties of oxidized and reduced [Fe4Se4] derivatives of Clostridium vinosum high-potential iron protein

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProton NMR spectra and electron-transfer properties of oxidized and reduced [Fe4Se4] derivatives of Clostridium vinosum high-potential iron proteinM. Sola, J. A. Cowan, and Harry B. GrayCite this: J. Am. Chem. Soc. 1989, 111, 17, 6627–6630Publication Date (Print):August 1, 1989Publication History Published online1 May 2002Published inissue 1 August 1989https://doi.org/10.1021/ja00199a023RIGHTS & PERMISSIONSArticle Views94Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (465 KB) Get e-Alerts Get e-Alerts

Electron paramagnetic resonance determination of a low-lying excited state in Chromatium vinosum high-potential iron protein

The temperature dependence of the EPR spectrum of oxidized high-potential iron protein from Chromatium vinosum has been studied. From line width and intensity measurements it is possible to determine the position of the first excited unoccupied state, 160 +/- 10 cm-1 above the ground state orbital.

The reaction of Euglena gracilis cytochrome c-552 with nonphysiological oxidants and reductants

The reaction of Euglena gracilis cytochrome c-552 (cytochrome f) with the nonphysiological reactants potassium ferrocyanide, potassium ferricyanide, sodium ascorbate, sodium dithionite, and Chromatium vinosum high potential nonheme iron protein was studied by stopped-flow and temperature-jump kinetic methods. The reaction of the purified, water-soluble protein with the reactants was investigated as a function of ionic strength, pH, and temperature. The results demonstrated that reduction and oxidation takes place at a negatively charged site on the cytochrome c-552 surface. Participation of specific amino acid residues in electron transfer is implicated from the pH results. The results obtained for the nonphysiological reactions of cytochrome c-552 are compared with available data for horse heart cytochrome c and Rhodospirillum rubrum cytochrome c 2. The results strongly suggest that Euglena gracilis cytochrome c-552 undergoes nonphysiological oxidation and reduction by a mechanism different from that found for cytochrome c or cytochrome c 2.

Iron-sulfur proteins: structural chemistry of their chromophores and related systems.

AbstractIntensive studies of iron‐sulfur proteins were begun only a decade or so ago but many biological and physicochemical data have since been accumulated and summarized[1–5]. As a result of the very recent X‐ray analyses of the structures of rubredoxin[6], ferredoxin (Peptococcus aerogenes)[7] and the Chromatium vinosum high potential iron protein (Hipip)[8], it has become possible to review our understanding of the nature and function of the inorganic chromophores in these proteins; to relate these findings to ‘model’ systems of varying relevance; but of more general interest, to comment on redox processes in biological systems particularly with respect to what might be termed electron transfer—allosteric effects in metalloenzymes.