High-potential Iron-sulfur Protein Research Articles

Characterization of a partially unfolded high potential iron protein.

A partially unfolded state of the Fe4S4-containing high potential iron-sulfur protein from Chromatium vinosum has been detected and characterized by NMR spectroscopy following addition of a concentrated solution of guanidinium chloride to the native protein. This intermediate species (i) maintains the polymetallic center, (ii) exhibits a largely collapsed secondary structure, and (iii) undergoes fast cluster decomposition upon oxidation. This information is framed into the knowledge about this class of proteins, and the possible role of this intermediate with respect to the in vivo folding/unfolding process is discussed as well its role in the slow hydrolytic degradation characteristic of oxidized HiPIPs.

Electron transfer from high-potential iron-sulfur protein and low-potential cytochrome c-551 to the primary donor of Rubrivivax gelatinosus reaction center mutationally devoid of the bound cytochrome subunit

The electron transfer reactions from low-potential cytochrome c-551, high-potential iron-sulfur protein (HiPIP) and cytochrome c′ to the photosynthetic reaction center mutationally devoid of the bound cytochrome subunit in the C244 mutant of the purple bacterium Rubrivivax gelatinosus were investigated using time-resolved optical spectroscopy. Cytochrome c-551 was the best electron donor. Both HiPIP and low-potential cytochrome c-551 can also react with the bound cytochrome subunit, but in this case HiPIP appeared to be more efficient than cytochrome c-551. Identification of soluble immediate electron donors to the bacteriochlorophyll dimer indicates that in Rvi. gelatinosus the bound cytochrome subunit is not essential for photosynthetic electron transport and growth. The presence of high- and low-potential electron carriers (HiPIP, E m=330 mV, and cytochrome c-551, E m=50 mV) involved in the reduction of the bacteriochlorophyll dimer and the bound cytochrome subunit suggests that two alternative electron transfer pathways to the photosynthetic reaction center may exist in Rvi. gelatinosus.

Cloning and sequencing of the gene encoding the high potential iron-sulfur protein (HiPIP) from the purple sulfur bacterium Chromatium vinosum

The gene encoding the high potential iron-sulfur protein (HiPIP) of Chromatium vinosum strain D (DSM 180 T) was cloned from an EcoRI- HindIII digest of genomic DNA. A nucleotide sequence of 648 bp length was determined which contained the coding region and putative promoter and termination sites. The gene codes for a 122 residue 12761 Da protein. The C-terminal 85 residues are those of the previously biochemically determined sequence, whereas the N-terminal 37 residues constitute a leader peptide which shows characteristics of the double arginine signal sequences of complex cofactor containing periplasmic proteins.

The Primary Structure of Rhodoferax fermentans High‐Potential Iron‐Sulfur Protein, an Electron Donor to the Photosynthetic Reaction Center

The complete amino acid sequence of Rhodoferax fermentans high-potential iron-sulfur protein (Hipip), which is known to be an efficient electron donor to the photosynthetic reaction center, has been determined using both N-terminal and C-terminal analyses. The sequence contains 75 residues, with 11 positive charges, 10 negative charges, and one histidine residue. The molecular mass of apo-Hipip, determined by electrospray ionization mass spectrometry, is 7849.64 Da. Multiple sequence alignment, based both on primary and tertiary structure information, reveals conservation of Tyr19 and Gly75 (Chromatium vinosum numbering) in addition to the four [Fe4S4]-bound cysteines. The Hipip from Rf. fermentans is most similar (57% similarity) to the Hipip from Rubrivivax gelatinosus, a photosynthetic bacterium belonging to the beta-1 subgroup of the proteobacteria.

ON THE ROLE OF CYTOCHROME C8 IN PHOTOSYNTHETIC ELECTRON TRANSFER OF THE PURPLE NON-SULFUR BACTERIUM RHODOFERAX FERMENTANS

We report on the isolation, purification and functional characterization of a soluble c-type cytochrome from light-grown cells of the purple phototroph Rhodoferax fermentans. This cytochrome is basic (pI = 8), has a molecular mass of 12 kDa, and is characterized by a midpoint reduction potential of +285 mV. Partial analysis of the N-terminus amino-acid sequence shows a high similarity with cytochromes of c8 type (formerly called Pseudomonas cytochrome c-551 type). Time-resolved spectrophotometric studies show that this cytochrome c8 reduces the tetraheme subunit of the photosynthetic reaction center, in a fast (sub-ms) and a slow (ms) phase. Competition experiments in the presence of both cytochrome c8 and high potential iron-sulfur protein (HiPIP), isolated from the same microorganism, show that cytochrome c8 oxidation is decreased upon addition of HiPIP. These observations suggest that cytochrome c8 and HiPIP might play alternative roles in the photosynthetic electron flow of Rhodoferax fermentans.

Copper(II) complexes of tetrapeptides containing cysteinyl and histidinyl residues

It is becoming increasingly apparent that the coordination chemistry of oligopeptides with Cys-X-Y-Cys and His-X-Y-Cys sequences (X, Y = variable amino acid residues) is an important theme in transition metal and bioinorganic chemistry(1,2). The Cys-X-Y-Cys sequence is often encountered in the metal binding site of several metalloproteins, such as iron–sulfur proteins, e.g. rubredoxins(3) and ferrodoxins(4), high potential iron–sulfur proteins(5), metallothioneins(6), zinc proteins(7) etc. The amino acid sequence His-X-Y-Cys is also of bioinorganic importance since it has been found in the active site of copper(8), iron–sulfur(9) and zinc(10) proteins. We are currently seeking to develop the area of transition metal/Cys-X-Y-Cys and His-X-Y-Cys peptide interactions(11,12); our goals are: (i) to elucidate the role of these sequences in the biological activity and mechanism of action of the above metallobiomolecules, and (ii) to find possible new and more effective agents for the elimination of heavy metals from the human organism or from contaminated waste waters. Here we report the preparation and preliminary characterization of copper(II) complexes of tetrapeptide ligands containing the Cys-X-Y-Cys and His-X-Y-Cys sequences.

Structural origins of redox potentials in Fe-S proteins: electrostatic potentials of crystal structures

Redox potentials often differ dramatically for homologous proteins that have identical redox centers. For two types of iron-sulfur proteins, the rubredoxins and the high-potential iron-sulfur proteins (HiPIPs), no structural explanations for these differences have been found. We calculated the classical electrostatic potential at the redox site using static crystal structures of four rubredoxins and four HiPIPs to identify important structural determinants of their redox potentials. The contributions from just the backbone and polar side chains are shown to explain major features of the experimental redox potentials. For instance, in the rubredoxins, the presence of Val 44 versus Ala 44 causes a backbone shift that explains a approximately 50 mV lower redox potential in one of the four rubredoxins. This result is consistent with experimental redox potentials of five additional rubredoxins with known sequence. Also, we attribute the unusually lower redox potentials of two of the HiPIPs studied to a less positive electrostatic environment around their redox sites. Finally, molecular dynamics simulations of solvent around static rubredoxin crystal structures indicate that water alone is a major factor in dampening the contribution of charged side chains, in accord with experiments showing that mutations of surface charges produce relatively little effect on redox potentials.

The solution structure refinement of the paramagnetic reduced high-potential iron-sulfur protein I from Ectothiorhodospira halophila by using stable isotope labeling and nuclear relaxation.

The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.

Synthesis of a new 4Fe4S cluster with a biomimetic bulky thiolate ligand as model for the [Fe 4S 4] [formula omitted] states of high potential iron-sulfur proteins

The new clusters (X) 2[Fe 4S 4(SCH 2tib 4] (XEt 4N, Ph 4P) have been synthesized with a sterically encumbered and benzyl-type thiolate ligand, in order to study its influence on the formation and stability of the oxidized state [Fe 4S 4] 3+. Both clusters exhibit a stable redox couple in CH 2Cl 2, and the oxidized (Ph 4P) [Fe 4S 4(SCH 2tib) 4] can be generated in solution by chemical oxidation. EPR and 1H NMR studies show that this species is unstable in solution at room temperature and that it decomposes, possibly by way of a transient [3Fe4S] + state. The isotropic shift, longitudinal relaxation time and temperature dependence of the SCH 2 protons' resonance have been analyzed both in the [4Fe4S] 2+ and [4Fe4S] 3+ states, and compared to results available on the same oxidation state centers in [4Fe4S] proteins.

Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.

The kinetics of photo-induced electrontransfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferarfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of 4.8 x 10(7) M(-1) x s(-1). The limiting value of kobs at high HiPIP concentration is 95 s(-1). The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is approximately 3 x 10(5) s(-1). This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 microM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 10(6) M(-1) x s(-1) and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferar fermentans HiPIP is a very effective electron donor to the photosynthetic RC.

The influence of a surface charge on the electronic and steric structure of a high potential iron-sulfur protein

The recombinant high-potential iron-sulfur protein (HiPIP) iso-I from Ectothiorhodospira halophila has been mutated at position 68. The αC of Val 68 is within a 0.6-nm sphere from the closest iron ion of the cluster. The valine residue has been replaced by a negatively charged glutamate residue (V68E) and by a positively charged lysine residue (V68K). With respect to the recombinant wild-type protein the reduction potentials of the V68E and V68K variants are –21±2 and +29±2 mV respectively (200 mM NaCl, pH 7, 25 °C). The solution structure of the V68E mutant was solved up to a pairwise RMSD of 66 pm for backbone atoms and 138 pm for all heavy atoms. The structure of the variant is very similar to that of recombinant wild type, indicating that the observed changes in reduction potentials are largely due to the effect of the introduced charges. It is proposed that the valence distribution within the oxidized iron-sulfur cluster is affected only slightly by the change in charge at position 68, but consistently with a simple electrostatic model.

1H NMR of high-potential iron-sulfur protein from the purple non-sulfurbacterium Rhodoferax fermentans.

Oxidized and reduced forms of high-potential iron-sulfur protein (HiPIP) from the purple non-sulfur photosynthetic bacterium Rhodoferax fermentans have been characterized using 1H-NMR spectroscopy. Pairwise and sequence-specific assignments of hyperfine-shifted 1H-NMR signals to protons of cysteine residues bound to the [4Fe-4S]3+/2+ cluster have been performed using one-dimensional NOE and exchange spectroscopy experiments. 1H-NMR hyperfine shifts and relaxation rates of cluster-bound Cys beta-CH2 protons indicate that in the [4Fe-4S]3+ cluster one iron ion can be formally described as Fe(III), while electron density corresponding to one electron is unevenly delocalized onto the remaining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys beta-CH2 protons in HiPIPs from six different sources as a function of the Fe-S-C beta-C alpha dihedral angle, indicate that the major contribution is due to a dipolar metal-centered mechanism, with a non-negligible contribution from a ligand-centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi-quantitative tool for extracting structural information from relaxation time measurements is proposed.

An exchange coupling model for the Fe 4S 43+ polymetallic center present in high potential iron-sulfur proteins

We present here a theoretical structural model for the Fe 4S 4 3+ polymetallic center, present in proteins and synthetic compounds, which under magnetic exchange coupling and double exchange coupling provides the best agreement between calculated and experimental properties. To test our approach we have fit the hyperfine coupling constants of the unpaired electrons with the 57Fe nuclei, the temperature dependence of the NMR contact shifts which are available for proteins, and the magnetic susceptibility data available for a synthetic compound. The model allows for either three or four different J values to start with, and a double exchange operator. Both the model and the fitting parameters are satisfactory from a chemical point of view.

A Serine → Cysteine Ligand Mutation in the High Potential Iron−Sulfur Protein from Chromatium vinosum Provides Insight into the Electronic Structure of the [4Fe−4S] Cluster

We have succeeded in preparing a mutant of the High Potential Iron−Sulfur Protein (HiPIP) from Chromatium vinosum in which a cysteine ligand has been replaced by a serine (C77S). Proton chemical shift data and nuclear Overhauser effects indicate that structural perturbations induced by the C77S mutation are minimal in both the oxidized and reduced forms of the HiPIP. The reduction potential of C77S is 25 mV lower than that of the wild-type HiPIP (WT) (0.2 M ionic strength, pH 4.5−9.0, 25 °C). Assignment of the hyperfine shifted signals in the 1H NMR spectrum of oxidized C77S revealed that the temperature dependences of the signals associated with cluster-ligating residues 46 and 77 are Curie and anti-Curie type, respectively, and are thus the reverse of those in WT. Taken together, these observations indicate that the iron bound to Ser-77 is less reducible than the corresponding iron in WT. The results are consistent with a previous model of the electronic structure of oxidized HiPIP clusters, confirming ...

Development of supramolecular metalloprotein mimics

Abstract

Characterization of an autoreduction pathway for the [Fe4S4]3+ cluster of mutant Chromatium vinosum high-potential iron proteins. Site-directed mutagenesis studies to probe the role of phenylalanine 66 in defining the stability of the [Fe4S4] center provide evidence for oxidative degradation via a [Fe3S4] cluster.

A number of point mutations of the conserved aromatic residue phenylalanine 66 (Phe66Tyr, -Asn, -Cys, -Ser) in Chromatium vinosum high-potential iron sulfur protein have been examined with the aim of understanding the functional role of this residue. Nonconservative replacements with polar residues have a minimal effect on the midpoint potential of the [Fe4S4]3+/2+ cluster, typically < +25 mV, with a maximum change of +40 mV for Phe66Asn. With the exception of the Phe66Tyr mutant, the oxidized state was found to be unstable relative to the recombinant native, with regeneration of the reduced state. The pathway for this transformation involves degradation of the cluster in a fraction of the sample, which provides the reducing equivalents required to bring about reduction of the remainder of the sample. This degradative reaction proceeds through a transient [Fe3S4]+ intermediate that is characterized by typical g values and power saturation behavior and is prompted by the increased solvent accessibility of the cluster core in the nonconservative Phe66 mutants as evidenced by 1H-15N HMQC NMR experiments. These results are consistent with a model where the critical role of the aromatic residues in the high-potential iron proteins is to protect the cluster from hydrolytic degradation in the oxidized state.

Rationalization of the reduction potentials within the series of the high potential iron-sulfur proteins

An analysis of the factors affecting reduction potentials within a series of high potential iron-sulfur proteins (HiPIP) has been performed by calculating the different contributions to the variation of electrostatic energy upon addition of one electron to the oxidized form of the protein. Molecular dynamics calculations were used to generate model structures of HiPIPs for which X-ray data are not available, starting from the known structures of highly homologous proteins. We have calculated and analyzed the contributions to the electrostatic energy deriving from the net charges present on the surface of the protein, the partial charges present on the uncharged residues, the polarizability of the protein atoms, the solvent dipoles and polarizabilities. A positive correlation with the reduction potentials was found only for the contribution due to the net charge of the protein which, in the absence of other factors such as differences in the coordination properties and in reorganizational energy upon reduction, is proposed to represent the determining effect for the large variation in reduction potential within this series of biological electron carriers.

The Solution Structure of Oxidized HiPIP I from Ectothiorhodospira halophila; Can NMR Spectroscopy Be Used to Probe Rearrangements Associated with Electron Transfer Processes?

AbstractIn the 1H NMR spectrum of the oxidized form of the high‐potential iron‐sulfur protein (HiPIPI) from Ectothiorhodospira halophila, 91% of the total proton resonances and 100% of the residues have been assigned. The standard COSY, NOESY, and TOCSY sequences have been optimized for the paramagnetism of the molecule. Extensive assignment of the 15N NMR spectrum has been obtained through HMQC spectra. With 1437 dipolar connectivities, of which about 10% involved fast‐relaxing protons, a family of 18 structures was generated with an RMSD of 0.65 Å by using the programs developed by Wüthrich. The family of structures was further refined by various calculation steps; the final RMSD was 0.48 Å. The structures appear to be very similar but not equal to the structures of the reduced protein. Despite the similarity in structure, significant variations in the chemical shifts are observed. A similar behavior was observed for the homologous protein from Chromatium vinosum. It is concluded that NMR is a sensitive tool to monitor differences between oxidized and reduced proteins; however, the detailed structural variations should be evaluated with caution at the present level of resolution, which roughly corresponds to a resolution of 2.5 Å in an X‐ray structure.

The role of a conserved tyrosine residue in high‐potential iron sulfur proteins

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

HiPIP oxido-reductase activity in membranes from aerobically grown cells of the facultative phototroph Rhodoferax fermentans

The role of the periplasmically located, water-soluble, HiPIP (high-potential iron-sulfur protein) in the respiratory chain of the facultative phototroph Rhodoferax fermentans has been examined. The oxidized HiPIP is reduced by succinate-dependent respiration via the bc 1 complex, this reaction being inhibited by myxothiazol and/or stigmatellin. The reduced HiPIP can be oxidized by the membrane-bound cytochrome oxidase, this reaction being inhibited by 0.1 mM cyanide. We conclude that aerobically grown Rf. fermentans contains a redox chain in which HiPIP mediates electron transfer between the bc 1 complex and the cb-type cytochrome oxidase.