Plant-type Ferredoxins Research Articles

The “Rieske” center in mitochondrial bc1 complex and in bacterial dioxygenases

Rieske type [2Fe-2S] clusters are present in the bc 1 complex of the respiratory chain as well as in bacterial dioxygenases. All these Rieske clusters are thought to have histidine ligands to the Fe II of the reduced complex as determined by ENDOR and ESEEM spectroscopy [1,2] but they differ in their redox potentials. We have compared the spectra and redox properties of the water soluble fragment of the Rieske center of bovine heart mitochondrial bc 1 complex (ISF; E m = + 3 0 0 mV) and of the ferredoxin of the benzene dioxygenase from Pseudomonas putida ML2 (Fdbed; E m = -150 mY). The redox potential of both proteins could be determined in solution by cyclic voltammetr~/ and by spectroelectrochemistry in an OTTLE cell. The redox potential of the ISF is pH dependent above pH = 7. From the fit of the pH dependence of the E m, two redox dependent pK a values of 7.6 and 9.2 on the oxidized protein were obtained [3]; the same pK values could also be deduced from the pH dependence of the CD spectrum [4]. These pK values are not observed in the dioxygenase Rieske clusters. The CD spectra of Rieske type clusters are significantly different from those of plant type ferredoxins. A strong negative CD band is present at 20 000 cm-' (500 rim) in all reduced Rieske clusters. Th s band could be assigned to the highest energy magnetically allowed d -~d transition (dz2 -~dxz) of the Fe II. This band is high!y indicative for the distortion of the tetrahedral symmetry of the Fe; in plant type ferredoxins having four cysteine ligands to the Fe, it occurs in the near infrared at 5800 cm-' [5].

2-Oxo-1,2-dihydroquinoline 8-Monooxygenase, a Two-component Enzyme System from Pseudomonas putida 86

2-Oxo-1,2-dihydroquinoline 8-monooxygenase, which catalyzes the NADH-dependent oxygenation of 2-oxo-1,2-dihydroquinoline to 8-hydroxy-2-oxo-1,2-dihydroquinoline, is the second enzyme in the quinoline degradation pathway of Pseudomonas putida 86. This enzyme system consists of two inducible protein components, which were purified, characterized, and identified as reductase and oxygenase. The yellow reductase is a monomeric iron-sulfur flavoprotein (M(r), 38,000), containing flavin adenine dinucleotide and plant-type ferredoxin [2Fe-2S]. It transferred electrons from NADH to the oxygenase or to some artificial electron acceptors. The red-brown oxygenase (M(r), 330,000) consists of six identical subunits (M(r), 55,000) and was identified as an iron-sulfur protein, possessing about six Rieske-type [2Fe-2S] clusters and additional iron. It was reduced by NADH plus catalytic amounts of reductase. For monooxygenase activity, reductase, oxygenase, NADH, molecular oxygen, and substrate were required. The activity was considerably enhanced by the addition of polyethylene glycol and Fe2+. 2-Oxo-1,2-dihydroquinoline 8-monooxygenase revealed a high substrate specificity toward 2-oxo-1,2-dihydroquinoline, since none of 25 other tested compounds was converted. Based on its physical, chemical, and catalytic properties, we presume 2-oxo-1,2-dihydroquinoline 8-monooxygenase to belong to the class IB multicomponent non-heme iron oxygenases.

Sulredoxin: a novel iron-sulfur protein of the thermoacidophilic archaeon Sulfolobus sp. strain 7 with a Rieske-type [2Fe-2S] center

A novel pink [2Fe-2S] protein has been purified from the cytosol fraction of the thermoacidophilic archaeon Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius 7) and called "sulredoxin." Its absorption, circular dichroism, and electron paramagnetic resonance spectra suggest the presence of a Rieske-type [2Fe-2S] cluster (g-factors of 2.01, 1.91, and 1.79; average g-factor [gav] = 1.90) which is remarkably similar to that of Thermus thermophilus respiratory Rieske FeS protein (J. A. Fee, K. L. Findling, T. Yoshida, R. Hille, G. E. Tarr, D. O. Hearshen, W. R. Dunham, E. P. Day, T. A. Kent, and E. Münck, J. Biol. Chem. 259:124-133, 1984) and distinctively different from those of the plant-type ferredoxins (gav = 1.96). Sulredoxin, which is the first Rieske-type [2Fe-2S] protein isolated from an archaeal species, does not function as an electron acceptor of the cognate 2-oxoacid:ferredoxin oxidoreductase. Whether sulredoxin is derived from the archaeal membrane-bound respiratory Rieske-type FeS center (gy = 1.91) is the subject of further investigation.

Differential activities of heterocyst ferredoxin, vegetative cell ferredoxin, and flavodoxin as electron carriers in nitrogen fixation and photosynthesis in Anabaena sp.

In cyanobacteria an increasing number of low potential electron carriers is found, but in most cases their contribution to metabolic pathways remains unclear. In this work, we compare recombinant plant-type ferredoxins from Anabaena sp. PCC 7120, encoded by the genes petF and fdxH, respectively, and flavodoxin from Anabaena sp. PCC 7119 as electron carriers in reconstituted in vitro assays with nitrogenase, Photosystem I, ferredoxin-NADP(+) reductase and pyruvate-ferredoxin oxidoreductase. In every experimental system only the heterocyst ferredoxin catalyzed an efficient electron transfer to nitrogenase while vegetative cell ferredoxin and flavodoxin were much less active. This implies that flavodoxin is not able to functionally replace heterocyst ferredoxin. When PFO-activity in heterocyst extracts was reconstituted under anaerobic conditions, both ferredoxins were more efficient than flavodoxin, which suggested that this PFO was of the ferredoxin dependent type. Flavodoxin, synthesized under iron limiting conditions, replaces PetF very efficiently in the electron transport from Photosystem I to NADP(+), using thylakoids from vegetative cells.

Recombinant expression of the fdxD gene of Rhodobacter capsulatus and characterization of its product, a [2Fe-2S] ferredoxin.

A gene called fdxD that could potentially code for a ferredoxin has recently been identified upstream of the nitrogenase structural genes in Rhodobacter capsulatus [Willison, Pierrard and Hübner (1993) Gene 133, 39-46]. In the present study, the fdxD gene product has been overproduced in Escherichia coli in a soluble form. The recombinant protein, pink in colour, was purified to homogeneity, and biochemically characterized as a new ferredoxin. It represents the fifth ferredoxin so far identified in R. capsulatus and was designated FdV. Its N-terminal sequence is identical with that of the native ferredoxin isolated from R. capsulatus. U.v-visible-absorption spectra as well as results of c.d. and e.p.r. spectroscopy demonstrated that the fdxD product contained a [2Fe-2S] cluster correctly assembled and incorporated into the polypeptide. Although similar to plant-type ferredoxins, FdV appeared poorly competent in the photo-reduction of NADP+. On the basis of in vitro assays, FdV cannot serve as an electron donor for nitrogenase. The lack of reactivity of FdV in either of these assays may primarily be due to its relatively high mid-point redox potential (E'o = -220 mV, pH 7.5).

Purification and properties of a bacterial-type ferredoxin from the nitrogen-fixing cyanobacterium Anabaena variabilis ATCC29413

Three soluble ferredoxins were purified to homogeneity from nitrogen-fixing cultures of Anabaena variabilis (ATCC 29413) and characterized. The purified proteins have different absorption spectra, molecular mass, iron content, amino-acid composition and resistance to O 2 inactivation. Two were plant-type ferredoxins FdI and FdxH, corresponding to the previously reported ferredoxins II and I (Böhme, H. and Schrautemeier, B. (1987) Biochim. Biophys. Acta 891, 1–7). The third ferredoxin (ferredoxin III) (previously not described in cyanobacteria) was a bacterial-type ferredoxin. Ferredoxin III has a molecular mass of about 6 kDa and contains 3–4 atoms Fe/mol. Native (oxidized) ferredoxin III shows an EPR-signal at g = 2.014 that disappears after reduction by dithionite, characteristic of ferredoxins containing three-iron clusters. Ferredoxin III, like ferredoxin FdxH, is inactivated by oxygen. Ferredoxin III supports higher rates of C 2H 2 reduction by Rhodobacter capsulatus nitrogenase than FdI and higher rates of H 2 evolution by clostridial hydrogenase than FdI and FdxH. Combined nitrogen supresses the synthesis of both nitrogenase and ferredoxin III. These data suggest a possible role of ferredoxin III (bacterial-type) in nitrogen fixation by A. variabilis.

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

The cyanobacterium Nostoc sp. strain PCC 73102, cultured under nitrogen-fixing conditions, was investigated for the occurrence of ferrodoxins by SDS-PAGE/Western immunoblots using antisera directed against both a major plant-type and a bacterial-type ferredoxin purified from Anabaena variabilis. Immunocytological labelling and transmission electron microscopy were used to study the distribution of both types of ferredoxins in the Nostoc cells. SDS-PAGE/Western immunoblots revealed two proteins/polypeptides in the Nostoc strain, immunologically related to two soluble ferredoxins purified from Anabaena variabilis: the major plant-type ferredoxin (Fd I) and a bacterial-type ferredoxin (Fd III). Immunolocalization showed a uniform distribution of the plant-type and the bacterial-type ferredoxin in both the photosynthetic vegetative cells and in the nitrogen-fixing heterocysts, with no specific association with any subcellular inclusions. Using the particle analysis of an image processor, the labelling associated with the vegetative cells, expressed as number of gold particles per cell area, was found to be only slightly higher (1.2x) or almost twice as high (1.9x) compared to the heterocysts for the major plant-type and the bacterial-type ferredoxin, respectively.

A synthetic analogue for the active site of plant-type ferredoxin: two different coordination isomers by a four-cys-containing [20]-peptide.

The (Fe2S2)2+ complex of an artificial 20-peptide ligand, Ac-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Pro-Leu-Leu-T hr-Cys- Val-NH2, containing an invariant Cys-A-B-C-D-Cys-X-Y-Cys (A, B, C, D, X, Y = amino acid residues) fragment of plant-type ferredoxins was synthesized by a ligand exchange method with [Fe2S2(S-t-Bu)4]2-. 1H-nmr spectroscopic and electrochemical data of the complex indicate the presence of two coordination isomers. One of them having a Cys-X-Y-Cys bridging coordination to the two Fe(III) ions, has the (Fe2S2)2+ core environment similar to those of the denatured plant-type ferredoxins and exhibits a positive shifted redox potential at -0.64 V vs saturated colonel electrode (SCE) in N,N-dimethylformamide (DMF). Another isomer with the Cys-A-B-C-D-Cys bridging coordination shows a negative redox potential at -0.96 V vs SCE in DMF.

PH-induced conformational changes in spinach ferredoxin: Steady-state and time-resolved fluorescence studies

Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I − and Cs +) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I −, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP + oxidoreductase and the rise in chloroplast stromal pH in the light.

Proton NMR spectra of vertebrate iron-sulfur [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)

A new [2Fe-2S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe-4S] ferredoxin in Escherichia coli.

A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156. The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing characteristic of [2Fe-2S] plant and cyanobacterial ferredoxins. The amino acid sequence was found to share approximately 25% amino acid similarity with plant-type ferredoxins. The gene was named fdxC. Expression of the fdxC and fdxN genes together in Escherichia coli was accomplished by subcloning the genes in the vector pUC18 downstream of the lac promoter. Cells containing this plasmid produced a red and a brown protein corresponding to the fdxC and fdxN gene products, respectively. EPR and UV-visible absorption spectroscopy confirmed that the FdxC protein contained a [2Fe-2S] cluster and the FdxN protein contained two [4Fe-4S] clusters and that the centers were correctly assembled and inserted in the ferredoxins expressed in E. coli. Transcription (Northern blot) analysis showed that the genes were transcribed only under nitrogen-limiting (nif-derepressing) growth conditions.

Electron spin-echo spectroscopy of the iron–sulphur clusters of xanthine oxidase from milk

Xanthine oxidase from milk contains two different [2Fe-2S] clusters, Fe-S(I) and Fe-S(II). The environment of each type of cluster has been examined by electron spin-echo envelope modulation (ESEEM) spectroscopy, where modulations were observed for the iron–sulphur clusters in the reduced state. The spectral contributions of the two clusters were distinguished (a) by poising the samples at redox potentials such that either Fe-S(II) was predominantly reduced, or both iron–sulphur clusters were fully reduced and (b) by measurements at their characteristic g factors. Spectra of both [2Fe-2S] clusters showed weak 14N hyperfine interactions, similar to those seen in other [2Fe-2S] proteins, which were attributed to nitrogens of the polypeptide chain; there was no evidence for histidine imidazole coordination. Spectra of samples exchanged into 2H2O gave evidence for exchangeable protons in close proximity to Fe–S clusters. By these criteria the environment of the [2Fe-2S] clusters in this complex protein is similar to those in the plant-type ferredoxins. In addition, ESEEM spectra of molybdenum (V) in the desulpho-inhibited form of the enzyme did not show modulations due to 14N.

In vitro analysis of polypeptide requirements of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600

An in vitro study of the multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600 was performed. Phenol-stimulated oxygen uptake from crude extracts was strictly dependent on the addition of NAD(P)H and Fe2+ to assay mixtures. Five of six polypeptides required for growth on phenol were necessary for in vitro activity. One of the polypeptides was purified to homogeneity and found to be a flavin adenine dinucleotide containing iron-sulfur protein with significant sequence homology, at the amino terminus, to plant-type ferredoxins. This component, as in other oxygenase systems, probably functions to transfer electrons from NAD(P)H to the iron-requiring oxygenase component. Phenol hydroxylase from this organism is thus markedly different from bacterial flavoprotein monooxygenases commonly used for hydroxylation of other phenolic compounds, but bears a number of similarities to multicomponent oxygenase systems for unactivated compounds.

ChemInform Abstract: Cysteine‐Containing Oligopeptide Model Complexes of Iron‐Sulfur Proteins

Publisher Summary The relationship between the structure of the peptide environment and active site chemical properties is of considerable interest in the recent investigations. This chapter presents some examples of such specific relationships, particularly in the case of iron–sulfur proteins, because this class of proteins is distributed widely in living organisms ranging from bacterial cells to mammals. The major function of the proteins is currently known to be electron transfer and the redox catalysis associated with it. Iron–sulfur clusters play a decisive role in these biological electron transfer functions and the binding of such clusters is considered. Extensive model studies on metal sulfide/thiolate complexes have already been performed. A typical structure of a water-soluble globular protein consists of hydrophilic amino acid residues outside and hydrophobic ones inside. The hydrophobic environments support various electrostatic interactions within the protein that plays a crucial role in the enzymatic reaction. The chapter also discusses chelating effects of peptide ligands, [2Fe–2S] plant-type ferredoxin peptide model complexes, and hydrophobic effect of peptide and related ligands. Specific peptides can also serve as mediators for electron passage, and for protection against dioxygen, water, and protons.

Protein-protein interactions and antigenic relationships between the components of 4-methoxybenzoate monooxygenase and of benzene 1,2-dioxygenase from Pseudomonas putida.

The investigations presented in this paper were performed on two enzyme systems from Pseudomonas putida: (a) 4-methoxybenzoate monooxygenase, consisting of a NADH: putidamonooxin oxidoreductase and putidamonooxin, the oxygen-activating component, and (b) benzene 1,2-dioxygenase, a three-component enzyme system with an NADH: ferredoxin oxidoreductase, functioning together with a plant-type ferredoxin as electron-transport chain, and an oxygen-activating component similar to putidamonooxin in its active sites. The influence of temperature, ionic strength, and pH on the activities of 4-methoxybenzoate monooxygenase and of NADH: putidamonooxin oxidoreductase were investigated. The studies revealed that the activity of 4-methoxybenzoate monooxygenase is determined by the behaviour of the reductase. Spectroscopic measurements showed that the interaction between the two components of 4-methoxybenzoate monooxygenase influences the optical-absorption behaviour of one or both components. As a criterion for the affinity between the two components of 4-methoxybenzoate monooxygenase, the Km value of the reductase for putidamonooxin was determined and found to be 31 +/- 11 microM. Antibodies against both components of 4-methoxybenzoate monooxygenase were obtained from rabbits. The antibodies against putidamonooxin inhibited the O-demethylation reaction (up to 80%) and also the reduction of putidamonooxin by the reductase (up to 40%). The antibodies against putidamonooxin did not interact with the oxygen-activating component of benzene 1,2-dioxygenase. The electron-transport chains of 4-methoxybenzoate monooxygenase and benzene 1,2-dioxygenase could not be replaced by one another without a complete loss of enzyme activity.

Main chain fold of a [2Fe-2S]ferredoxin I from Aphanothece sacrum at 2.5 A resolution.

Crystal structure analysis of a [2Fe-2S]ferredoxin I from Aphanothece sacrum, a blue green alga, was carried out at 2.5 A resolution. The phase angle of each reflection was determined by the single isomorphous replacement method coupled with the anomalous dispersion effect for the uranium derivative. The four molecules in an asymmetric unit were clearly seen in a 3.9 A electron density map. The main chains of three molecules were traced at 2.5 A resolution. The structure of the remaining one molecule, however, remains unknown because of the poor electron density of the corresponding region. The three main chain folds exhibit the same topology as that in Spirulina platensis. The structural similarity between A. sacrum and S. platensis ferredoxins, whose amino acid sequences are different from each other by about 30%, strongly suggests that all plant-type ferredoxins have the same main chain fold.

Mössbauer spectroscopic studies of the terminal dioxygenase protein of benzene dioxygenase from Pseudomonas putida.

Mössbauer spectra obtained from the terminal dioxygenase protein of the benzene dioxygenase system from Pseudomonas putida show that it contains [2Fe--2S] centres similar to those of the two-iron plant-type ferredoxins. In the oxidized form the two iron atoms within the centre are high-spin ferric but with considerable inequivalence. In the reduced form the centre contains one extra electron, and this is localized on one of the iron atoms, which becomes high-spin ferrous.

The amino acid sequence of ferredoxin from Brassica napus (rape).

The amino acid sequence of the ferredoxin of Brassica napus was determined by using a Beckman 890C sequencer in combination with the characterization of peptides obtained by tryptic and chymotryptic digestion of the protein; some peptides were subdigested with thermolysin. The molecule consists of a single polypeptide chain of 96 amino acid residues and has an unblocked N-terminus. The primary structure shows considerable similarity with other plant-type ferredoxins.

The amino acid sequence of ferredoxin from Triticum aestivum (wheat).

The amino acid sequence of the ferredoxin of Triticum aestivum (wheat) was determined by using a Beckman 890C sequencer in combination with the dansyl--phenylisothiocyanate method to characterize peptides obtained by tryptic, chymotryptic and thermolytic digestion of CNBr-cleavage fragments. The molecule consists of a single polypeptide chain of 97 residues and has an unblocked N-terminus. It shows considerable similarity to other plant-type ferredoxins.