Primary structure of a high potential iron-sulfur protein from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas gelatinosa.

The third amino acid sequence of a high potential iron-sulfur protein, that of the non-sulfur purple photosynthetic bacterium Rhodopseudomonas gelatinosa, has been determined. It consists of a single polypeptide chain of 74 amino acid residues, which is slightly smaller than the high potential iron-sulfur proteins from the sulfur purple bacteria Chromatium vinosum (85 residues) and Thiocapsa pfennigii (81 residues). The sequence of the gelatinosa protein is similar to the C. vinosum and T. pfennigii proteins with 38% and 37% identically matching residues, although six gaps are proposed for the comparison (the C. vinosum and T. pfennigii proteins have 44% identically matching residues out of 73 positions compared with only one 4-residue gap). Only 17 redisues, including the 4 cystein residues essential for binding the four-iron-sulfur chromophore, are invariant in the three known sequences. A discussion of the role of conserved residues in maintenance of the three-dimensional structure and in electron transport is presented.     

Investigation of the role of a surface patch in the self-association of Chromatium vinosum high potential iron-sulfur protein.

The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear magnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g(z) region of the electron paramagnetic resonance (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flattened, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by this patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP.     

The high potential iron-sulfur cluster of aconitase is a binuclear iron-sulfur cluster.

It has been reported (Ruzicka, F.J., and Beinert, H. (1978) J. Biol. Chem. 253, 2514-2517) that aconitase in the oxidized state, as isolated, shows an electron paramagnetic resonance signal centered at g = 2.01, typical of high potential iron-sulfur proteins. Since the magnetic state corresponding to this signal has thus far only been found in tetranuclear iron-sulfur clusters in model compounds and proteins, it could be expected that aconitase also contains a [4Fe-4S] cluster. We show here that core extrusion, in the presence of hexamethylphosphoramide and o-xylyl-alpha,alpha'-dithiol and subsequent ligand exchange with p-trifluoromethylbenzenethiol yield absorption spectra typical of binuclear iron-sulfur clusters. According to the absorbance measured, the concentration of the extruded [2Fe-2S] cluster quantitatively accounts for the iron-sulfur content of the preparations examined. Preliminary studies of the 19F nuclear magnetic resonance spectrum obtained on extrusion with p-trifluoromethylbenzenethiol confirm the presence of a binuclear cluster in aconitase.     

The molecular structure of the high potential iron-sulfur protein isolated from Ectothiorhodospira halophila determined at 2.5-A resolution

The molecular structure of a high potential iron-sulfur protein (HiPIP) isolated from the purple photosynthetic bacterium, Ectothiorhodospira halophila strain BN9626, has been solved by x-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R value of 18.4% including all measured x-ray data from 30.0- to 2.5-A resolution. Crystals used in the investigation contained two molecules/asymmetric unit and belonged to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. An interpretable electron density map, obtained by combining x-ray data from one isomorphous heavy atom derivative with non-crystallographic symmetry averaging and solvent flattening, clearly showed that this high potential iron-sulfur protein contains 71 amino acid residues, rather than 70 as originally reported. As in other bacterial ferredoxins, the [4Fe-4S] cluster adopts a cubane-like conformation and is ligated to the protein via four cysteinyl sulfur ligands. The overall secondary structure of the E. halophila HiPIP is characterized by a series of Type I and Type II turns allowing the polypeptide chain to wrap around the [4Fe-4S] prosthetic group. The hydrogen bonding pattern around the cluster is nearly identical to that originally observed in the 85-amino acid residue Chromatium vinosum HiPIP and consequently, the 240 mV difference in redox potentials between these two proteins cannot be simply attributed to hydrogen bonding patterns alone.     

Biochemical and EPR characterization of a high potential iron-sulfur protein in Thiobacillus ferrooxidans

Abstract A soluble acid-stable high potential iron-sulfur protein (HiPIP) was purified from Thiobacillus ferrooxidans using the periplasmic extraction method. It was isolated in the form of a tetramer consisting of four subunits with a molecular mass of 5582 Da, and its biochemical and biophysical properties were characterized. The N-terminal amino acid sequence (15 residues) was compared with the nucleotide sequence of the iro gene isolated from another strain and the two sequences were found to be identical. The iron content measurement together with optical and EPR spectroscopic studies of the purified protein were consistent with the presence of one [4Fe-4S] cluster per subunit. The EPR spectrum recorded in the oxidized state was attributed to a [4Fe-4S]³⁺ cluster and the redox potential has been determined to be +380 mV.     

Oxidation-reduction properties of Chromatium vinosum high potential iron-sulfur protein.

The oxidation-reduction properties of the high potential iron-sulfur protein (HIPIP) from Chromatium vinosum have been investigated. Both equilibrium and kinetic measurements demonstrate electron transport by HIPIP is pH independent in the pH range 7-11. The kinetics of reduction (potassium ferrocyanide, SO2, S2O42-, sodium ascorbate, and Rhodospirillum rubrum cytochrome c2) and oxidation (potassium ferricyanide and Rhodospirillium rubrum cytochrome c2) of HIPIP are reported. Based on the data obtained with different reactants and the influence of ionic strength, pH, and temperature on the kinetics of oxidation and reduction, a number of conclusions can be drawn. (1) HIPIP undergoes rapid outer-sphere electron transfer with no evidence of kinetic complexity and no indication of complex formation with various reactants. (2) The site of oxidation of reduced HIPIP has an apparent negative charge while the site of reduction of oxidized HIPIP is uncharged. (3) HIPIP appears to interact with a physiological reactant (R. rubrum cytochrome c2) at the same site as nonphysiological oxidants or reductants suggesting single minimum energy pathways for the oxidation and reduction processes. (4) Based on a comparison of the rates of oxidation and reduction with different reactants, it appears that steric restrictions and differences in oxidation-reduction potential are less important than electrostatic attraction and/or repulsion in determining the absolute rate constants. (5) The thermodynamic activation parameters indicate that both oxidation and reduction by the iron hexacyanides are driven entropically with the enthalpic terms making no contribution to HIPIP oxidation and a small contribution to HIPIP reduction. Based on the data reported here and available structural and physical-chemical information, possible mechanisms of the oxidation and reduction of HIPIP are discussed and their relative merits analyzed. The more likely mechanisms include electron transfer via a tyrosine residue, electron transfer through a nonaqueous media to the iron-sulfur chromophore, and direct interaction between the iron-sulfur chromophore and the different oxidants and reductants.     

Expression and characterization of recombinant Rhodocyclus tenuis high potential iron-sulfur protein

The high potential iron-sulfur protein (HiPIP) from Rhodocyclus tenuis strain 2761 has been overproduced in Escherichia coli from its structural gene, purified to apparent homogeneity, and then characterized by an array of methods. UV-visible spectra of the reduced and oxidized recombinant protein were similar to those of the native protein. EPR of the oxidized protein shows g values of 2. 11, 2.03, and 2.03. ESI-MS gave a mass difference of 350 Da between the holoprotein and acid-treated protein, consistent with incorporation of a [Fe(4)S(4)] cluster in the holoprotein. The observed mass of the apoprotein was 6296.6 Da compared to the expected average molecular mass of 6297.2 Da of the apoprotein. The reduction potential was determined using cyclic and square-wave voltammetry to be 321 and 314 mV versus NHE, respectively. All the observed properties of the recombinant protein parallel those of the native protein or those of native HiPIPs in general, indicating correct folding and incorporation of the iron-sulfur cluster.     

Crystallization of a high potential iron-sulfur protein from the halophilic phototrophic bacterium Ectothiorhodospira halophila

Crystals of the high-potential iron-sulfur protein from Ectothiorhodospira halophila strain BN 9626 have been grown from 3.4 to 3.5 M ammonium sulfate solutions at pH 7.5. The crystals belong to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. There are 2 molecules/asymmetric unit. The crystals diffract to at least 1.8 A, are stable in the x-ray beam, and are suitable for a high resolution x-ray crystallographic analysis.     

Solution structure of the iron-sulfur cluster cochaperone HscB and its binding surface for the iron-sulfur assembly scaffold protein IscU

The interaction between IscU and HscB is critical for successful assembly of iron-sulfur clusters. NMR experiments were performed on HscB to investigate which of its residues might be part of the IscU binding surface. Residual dipolar couplings ( (1) D HN and (1) D CalphaHalpha) indicated that the crystal structure of HscB [Cupp-Vickery, J. R., and Vickery, L. E. (2000) Crystal structure of Hsc20, a J-type cochaperone from Escherichia coli, J. Mol. Biol. 304, 835-845] faithfully represents its solution state. NMR relaxation rates ( (15)N R 1, R 2) and (1)H- (15)N heteronuclear NOE values indicated that HscB is rigid along its entire backbone except for three short regions which exhibit flexibility on a fast time scale. Changes in the NMR spectrum of HscB upon addition of IscU mapped to the J-domain/C-domain interface, the interdomain linker, and the C-domain. Sequence conservation is low in the interface and in the linker, and NMR changes observed for these residues likely result from indirect effects of IscU binding. NMR changes observed in the conserved patch of residues in the C-domain (L92, M93, L96, E97, E100, E104, and F153) were suggestive of a direct interaction with IscU. To test this, we replaced several of these residues with alanine and assayed for the ability of HscB to interact with IscU and to stimulate HscA ATPase activity. HscB(L92A,M93A,F153A) and HscB(E97A,E100A,E104A) both showed decreased binding affinity for IscU; the (L92A,M93A,F153A) substitution also strongly perturbed the allosteric interaction within the HscA.IscU.HscB ternary complex. We propose that the conserved patch in the C-domain of HscB is the principal binding site for IscU.     

The solution structure of desulforedoxin,a simple iron-sulfur protein

Desulforedoxin is a simple dimeric protein isolated from Desulfovibrio gigas containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9?kDa with a 36-amino-acid monomer). ¹H NMR spectra of the oxidized Dx(Fe³⁺) and reduced Dx(Fe²⁺) forms were analyzed. The spectra show substantial line broadening due to the paramagnetism of iron. However, very low-field-shifted resonances, assigned to Hβ protons, were observed in the reduced state and their temperature dependence analyzed. The active site of Dx was reconstituted with zinc, and its solution structure was determined using 2D NMR methods. This diamagnetic form gave high-resolution NMR data enabling the identification of all the amino acid spin systems. Sequential assignment and the determination of secondary structural elements was attempted using 2D NOESY experiments. However, because of the symmetrical dimer nature of the protein standard, NMR sequential assignment methods could not resolve all cross peaks due to inter- and intra-chain effects. The X-ray structure enabled the spatial relationship between the monomers to be obtained, and resolved the assignment problems. Secondary structural features could be identified from the NMR data; an antiparallel β-sheet running from D5 to V18 with a well-defined β-turn around cysteines C9 and C12. The section G22 to T25 is poorly defined by the NMR data and is followed by a turn around V27-C29. The C-terminus ends up near residues V6 and Y7. Distance geometry (DG) calculations allowed families of structures to be generated from the NMR data. A family of structures with a low target function violation for the Dx monomer and dimer were found to have secondary structural elements identical to those seen in the X-ray structure. The amide protons for G4, D5, G13, L11 NH and Q14 NHε amide protons, H-bonded in the X-ray structure, were not seen by NMR as slowly exchanging, while structural disorder at the N-terminus, for the backbone at E10 and for the section G22–T25, was observed. Comparison between the Fe and Zn forms of Dx suggests that metal substitution does not have an effect on the structure of the protein.     

The soluble "high potential" type iron-sulfur protein from mitochondria is aconitase.

Properties of soluble high potential type iron-sulfur protein (HiPIP) from beef heart mitochondria were compared to those of aconitase from pig heart. The two proteins when purified to homogeneity by the criteria of sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis show identical light absorption characteristics. EPR signals of the HiPIP type centered at g = 2.01 when oxidized, isoelectric points at pH 8.5 to 8.6, are inseparable by SDS-polyacrylamide electrophoresis, and exhibit aconitase activity when activated by reducing agents in the presence of ferrous iron. The requirement for activation goes parallel to the intensity of the signal from the oxidized iron-sulfur cluster, i.e. the cluster is reduced in the active enzyme. We conclude that the soluble mitochondrial HiPIP is identical with aconitase. The relationships of iron to labile sulfide, molecular weight and unpaired spins in the EPR signal, and implications of our findings for the role of iron in aconitase are discussed.     

Crystallization and preliminary analysis of crystals of high potential iron-sulfur protein from Rhodospirillum tenue

Large single crystals of the high potential iron-sulfur protein isolated from Rhodospirillum tenue strain 3761 have been obtained. They belong to the space group P2(1) with unit cell dimensions of a = 36.7 A, b = 52.6 A, c = 27.6 A, and beta = 90.8 degrees. There are two molecules in the asymmetric unit. Based on oscillation photographs, the crystals diffract to at least 1.6 A resolution. They are stable in the x-ray beam and appear suitable for a high resolution x-ray structure analysis.     

The role of histidine-42 in the oxidation-reduction mechanism of Chromatium vinosum high potential iron-sulfur protein

The second order rate constants for the oxidation of high potential iron-sulfur protein (Hipip) of Chromatium vinosum by ferricyanide were determined as a function of ionic strength and pH. From the ionic strength results, calculations were done to correct the rate constant at each pH for the electrostatic interactions between Hipip and ferricyanide. The electrostatic corrections are necessary since the charge of the protein changes as a function of pH and can mask the ionization of mechanistically important amino acid residues. An apparent pK_a 7 was obtained from electrostatically corrected rate-pH profile, indicating the possible participation of histidine. Perturbation difference spectroscopic studies of Hipip as a function of pH also gave apparent pK_a values of 6.9 and 6.7 for the reduced and oxidized protein, respectively. That it was indeed His 42 (the only His in the polypeptide) that was responsible for the kinetic and spectroscopic pK_a values was demonstrated by modification of His 42 of Hipip by the histidine selective reagent diethylpyrocarbonate. No modification of Tyr 19 could be detected. It is concluded that either deprotonation or modification of His 42 results in the destabilization of the reduced cluster and thus a faster rate of oxidation. This work provides the first experimental evidence of the ‘squeeze effect’ mechanism (Carter, C.W., Jr., Kraut, J., Freer, S.T. and Alden, R.A. (1974) J. Biol. Chem. 249, 6339–6346) in which the polypeptide directly modulates the stability of the iron-sulfur cluster.     

Evidence for antiferromagnetic exchange coupling in the tetranuclear high potential iron-sulfur protein of Rhodopseudomonas gelatinosa.

EPR spectra of oxidized $\text{R. gelatinosa}$ HiPIP demonstrate two kinds of temperature dependent changes which can be analyzed in terms of an excited state at 142 ± 10cm^?1 and a second excited state at 490 ± 100cm^?1. These states represent further verification of antiferromagnetic exchange among the 4 irons in this tetranuclear cluster, with a value for the coupling constant of J = ?44cm^?1. Aside from resonance Raman spectroscopic results, this is the first report of a ladder of excited states predicted for exchange coupled ions.     

The high potential iron-sulfur center in Escherichia coli fumarate reductase is a three-iron cluster

The fumarate reductase complex and soluble enzyme from Escherichia coli have been investigated by low temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies. The results confirm the presence of one [2Fe-2S] cluster and show that the high potential iron-sulfur center is a 3Fe cluster of the type found in bacterial ferredoxins. Since the 3Fe cluster is present in catalytically competent enzyme and does not appear to be involved in any type of cluster conversion under reducing conditions, we conclude that it is an intrinsic component of the functional enzyme. The significance of the results is discussed in relation to the published amino acid sequence and the iron-sulfur cluster composition of bacterial fumarate reductases.     

Two distinct binding sites for high potential iron-sulfur protein and cytochrome c on the reaction center-bound cytochrome of Rubrivivax gelatinosus

The photosynthetic cyclic electron transfer of the purple bacterium Rubrivivax gelatinosus, involving the cytochrome bc(1) complex and the reaction center, can be carried out via two pathways. A high potential iron-sulfur protein (HiPIP) acts as the in vivo periplasmic electron donor to the reaction center (RC)-bound cytochrome when cells are grown under anaerobic conditions in the light, while cytochrome c is the soluble electron carrier for cells grown under (8)aerobic conditions in the dark. A spontaneous reversion of R. gelatinosus C244, a defective mutant in synthesis of the RC-bound cytochrome by insertion of a Km(r) cassette leading to gene disruption with a slow growth rate, restores the normal photosynthetic growth. This revertant, designated C244-P1, lost the Km(r) cassette but synthesized a RC-bound cytochrome with an external 77-amino acid insertion derived from the cassette. We characterized the RC-bound cytochrome of this mutant by EPR, time-resolved optical spectroscopy, and structural analysis. We also investigated the in vivo electron transfer rates between the two soluble electron donors and this RC-bound cytochrome. Our results demonstrated that the C244-P1 RC-bound cytochrome is still able to receive electrons from HiPIP, but it is no longer reducible by cytochrome c(8). Combining these experimental and theoretical protein-protein docking results, we conclude that cytochrome c(8) and HiPIP bind the RC-bound cytochrome at two distinct but partially overlapping sites.     

The influence of surface charge on the kinetics of ion-translocation across biological membranes

Rate equations have been developed which describe the concentration dependence for ion-translocation across charged membranes for those cases in which the translocation process can be considered to be formally equivalent with an enzymic process of a Michaelis-Menten type. We have limited ourselves to those cases in which the ion-translocational step through the membrane is electroneutral. In addition it is assumed that the sites on the membrane involved in the ion-translocation process can not move through the membrane when these sites are not occupied by ions.It is shown that in general deviations from Michaelis-Menten kinetics may be expected. In case of monovalent ion-translocation across oppositely charged membranes apparent negative homotrope cooperative effects may occur, whereas for ion-translocation across equally charged membranes apparent positive homotrope cooperative effects may be found. When the bulk aqueous phase also contains polyvalent ions both types of effects may occur both in the case of ion-translocation across oppositely charged membranes as well as with ion-translocation across a membrane of which the sign of the surface charge is the same as that of the ion translocated.Under limited conditions, also apparent single Michaelis-Menten kinetics may be observed. In these cases, however, the apparent K_m generally is no linear function of the concentration of a competing ion. It is shown that even when an ion does not bind to the translocation sites the K_m is affected by increasing concentrations of this ion, a phenomenon which is not expected when the membrane is not charged. The effects of divalent ions, added to the bulk aqueous phase as 1-1-electrolytes, upon the K_m are discussed in connection with in literature reported effects of Ca⁺⁺ upon the rate of uptake of several monovalent ions into plant cells.