Spinach ferredoxin

FIGURE 9.2 EPR powder pattern of the [2Fe-2S]1+ cluster in spinach ferredoxin. Trace A shows an attempt to fit the spectrum with the diagonal linewidth Equation 9.1. In trace B the spectrum is fitted with the nondiagonal g-strain Equation 9.18. Trace C shows an experiment in which the spectral features are slightly shifted (solid trace) under the influence of an external hydrostatic stress. (Data replotted from Hagen and Albracht 1982.)... 

FIGURE 9.3 Linewidth as a function of microwave frequency. The linewidth (FWHH) of the low-field gj-feature is plotted versus the frequency in L-, S-, X-, and Q-band. The left-hand panel is for the ferric low-spin heme in horse heart cytochrome c, and the right-hand panel is for the [2Fe-2S] cluster in spinach ferredoxin. (Data from Hagen 1989.)... 

Figure 20 Cyclic voltammograms recorded at an edge-oriented pyrolitic graphite electrode in an aqueous solution (pH 8) of spinach ferredoxin. In the absence (a) and in the presence (b) of [Cr(NH3)6J3 +. Scan rate 0.02 V s ...

Staples CR, Gaymard E, Stritt-Etter A-L, et al. 1998. Role of the 6484] cluster in mediating disulfide reduction in spinach ferredoxin thioredoxin reductase. Biochemistry 37 4612-20. 

Figure 5 300 MHz NMR spectrum of reduced spinach ferredoxin in 0.5 M Tris-HCl buffer, pH 7.4, 297 K (o) signals from residual oxidized protein (x) signals from impurities. A scheme of the dimetallic cluster in the ferredoxin with the proposed...

Plant type ferredoxins. Tagawa and Arnon (16) described the isolation of a ferredoxin from spinach chloroplast. This ferredoxin is a protein of 12,000 molecular weight, and consists of 97 amino acids (17). Spinach ferredoxin has abosrbance maxima at 325, 420 and 465 nm (18). Ferredoxins of this type have been isolated from other sources of plants and algae, e.g., alfalfa (19), taro (20), Leuceana glauca (21) and Scenedesmus (22). The prot s of thes erredoxins are similar in their properties to ferredoxin from spinach. 

The biological functions of chloroplast ferredoxins are to mediate electron transport in the photosynthetic reaction. These ferredoxins receive electrons from light-excited chlorophyll, and reduce NADP in the presence of ferredoxin-NADPH reductase (23). Another function of chloroplast ferredoxins is the formation oT" ATP in oxygen-evolving noncyclic photophosphorylation (24). With respect to the photoreduction of NADP, it is known that microbial ferredoxins from C. pasteurianum (16) are capable of replacing the spinach ferredoxin, indicating the functional similarities of ferredoxins from completely different sources. The functions of chloroplast ferredoxins in photosynthesis and the properties of these ferredoxin proteins have been reviewed in detail by Orme-Johnson (2), Buchanan and Arnon (3), Bishop (25), and Yocum et al. ( ). 

The 2Fe2S (S, acid-labile sulfur) ferredoxins have a redox active binuclear center, with each of the two iron atoms attached to the protein by two cysteinyl sulfur ligands and connected by two inorganic acid-labile sulfur ligands. At cty-ogenic temperatures these clusters are EPR detectable, with characteristic features in the vicinity of g = 1.94. Spinach ferredoxin has principal g values of 2.03, 1.96, and 1.88 and a broad absorbance spectrum with a weak maximum around 420 nm, giving these proteins a reddish brown color which bleaches on reduction. Ferredoxins are low potential electron carriers chloroplast ferredoxins function in photosynthetic electron transfer, but related proteins such as adrenal ferredoxin are involved in steroidogenic electron transfer in mitochondria in tissues which produce steroid hormones. 

Fig. 6. Electron paramagnetic resonance signal showing the g= 1.94 characteristic of the dithionite-reduced spinach ferredoxin, a plant-type iron-sulfur protein. Spectrum taken at 20 °K...

Bearden and Moss (12) and Moss et al. (152) presented the Mossbauer spectra of spinach ferredoxin in its oxidized and reduced states. These spectra showed the two iron atoms in the oxidized protein in identical electronic environments. Upon protein reduction, one of the iron atoms exhibited a spectrum characteristic of a high-spin ferrous ion. The... 

Mossbauer spectra of the reduced proteins in the above study are not consistent with subsequent data for these proteins (153,164,165). It is now believed (personal communications, W. H. Orme-Johnson and Graham Palmer) that 1, the samples in these experiments were impure, and 2. the buffers used in these experiments were not strong enough to maintain a buffer pH level during the dithionite reductions. Therefore, the Mossbauer spectra of reduced spinach ferredoxin in the above experiment resulted from a mixture of oxodized protein iron and iron from denatured protein material. 

Fig. 7. Mossbauer spectra of oxidized plant-type iron-sulfur proteins in zero applied magnetic field. Abbreviations AZI = A zotobacter Fe-S protein I, 4.6°K AZII = Azoiobacter Fe-S protein II, 4.2 °K Put. = Putidaredoxin, 4.2 °K Ad.— Pig Ad-renodoxin, 4.2 °K Clos. = Clostridial paramagnetic protein, 4.2 °K PPNR = Spinach ferredoxin, 4.5 °K Parsley = Parsley Ferredoxin, 4.2 °K. Velocity scale is relative to iron in platinum...

Fig. 8. Mossbauer spectra of oxidized plant-type iron-sulfur proteins in high applied magnetic field. Abbreviations Ad. = Pig Adrenodoxin, 4.2 °K, 46 kG PPNR = Spinach Ferredoxin, 4.5 °K, 50 kG Clos. = Clostridial Paramagnetic Protein, 4.2 °K, 46 kG AZI = Azotobacter Fe-S Protein I, 4.6°K, 46 kG AZII = Azotobacter Fe-S Protein II, 4.2 °K, 46 kG. Applied magnetic field is parallel to gamma-ray direction...

The Mossbauer spectra of spinach ferredoxin at 256 °K is shown in Fig. 9a the solid line on these spectra is the result of computer-simulated Mossbauer spectra. A magnetic field of 46 kilogauss parallel to the gamma-... 

Fig. 9. Mossbauer spectra and computed Mossbauer spectra for reduced spinach ferredoxin at 256 °K. (a) Lyophilized spinach ferredoxin in zero magnetic field (b) Lyophilized spinach ferredoxin with 46 kG magnetic field parallel to gamma-ray direction. Velocities relative to platinum source...

These spectra, taken at variable temperatures and a small polarizing applied magnetic field, show a temperature-dependent transition for spinach ferredoxin. As the temperature is lowered, the effects of an internal magnetic field on the Mossbauer spectra become more distinct until they result at around 30 °K, in a spectrum which is characteristic of the low temperature data of the plant-type ferredoxins (Fig. 11). We attribute this transition in the spectra to spin-lattice relaxation effects. This conclusion is preferred over a spin-spin mechanism as the transition was identical for both the lyophilized and 10 mM aqueous solution samples. Thus, the variable temperature data for reduced spinach ferredoxin indicate that the electron-spin relaxation time is around 10-7 seconds at 50 °K. The temperature at which this transition in the Mossbauer spectra is half-complete is estimated to be the following spinach ferredoxin, 50 K parsley ferredoxin, 60 °K adrenodoxin, putidaredoxin, Clostridium. and Axotobacter iron-sulfur proteins, 100 °K. 

The Mossbauer spectra of the reduced proteins at 4.2 °K are shown in Fig. 11 for 3.4 kilogauss applied field and in Fig. 12 for 46 kilogauss applied field. Since the spectra are so similar, we shall speak exclusively in terms of the spinach ferredoxin data. Fig. 13 is low-temperature... 

Fig. 10. Mossbauer spectra taken at various temperatures between 4.3 °K and 253 °K for lyophilized spinach ferredoxin with 580 G magnetic field applied parallel to the gamma-ray direction. Velocity scale relative to platinum- source...

Using these "improved parameters for site 1, the trial and error approach was then resumed in order to find a best fit for the site 2 parameters. Subsequently, the ENDOR values for the hyperfine interaction at site 2 were also obtained by Sands and his co-workers (160). Since these values were also in agreement with our own, the final parameters for spinach ferredoxin shown in Table 6 incorporate the combined effort of ENDOR and Mossbauer results, although the ENDOR results give no... 

Since gi arises from an S-state ion, spin-orbit interactions are not allowed to first order (163) and gi can therefore be assumed to be isotropic. It is assumed to be 2.019 in accord with the measurements of Title (166). With this assumption, the g-values for the ferrous iron can be derived using the above equation and the measured g-values for the proteins (Table 6). For spinach ferredoxin, these calculated values are g%x = 2.12, g%y — 2.07, and 221 = 2.00. 

The protein sequence data in Table 2 show that the cysteine residues in all the proteins occur in identical positions (18, 39, 44, 47, 77) in the sequence. Thus, the ligand field produced by the cysteinyl-sulfur atoms is not likely to be different among these proteins unless there is a difference in protein conformation which causes a displacement in one or more of the cysteinyl sulfur atoms. Note that a displacement of any cysteinyl sulfur atom in the model in Fig. 15 results in rhombic distortion at the iron to which it is ligated. Since, according to the spin-coupled model, this rhombic distortion will manifest itself in the difference between gx and gx for a particular protein, the EPR data in Table 1 provide a measure of the rhombic distortion around the ferrous iron in the reduced proteins. In particular, the g-values of adrenodoxin are axially symmetric while the g-values of spinach ferredoxin show a rhombic distortion. Thus, the observation of Kimura et al. (168) that adrenodoxin and spinach ferredoxin have different protein conformations is consistent with the prediction of the above model. 

Figure 4.6 Hydrogen bonding in functional nests (a) oxyanion hole in serine protease binding the substrate tetrahedral intermediate, (b) P-loop showing the p-phosphate (middle phosphate residue) of GTP bound to a compound nest, (c) Ca2+ binding in calmodulin. The calcium is bound to three carboxylate oxygen atoms which are in turn bound to bound to a five-amide compound nest (d) the cysteine-bound Fe2 unit in spinach ferredoxin.

S Nakamura. Initiation of sulfite oxidation by spinach ferredoxin-NADP reductase and ferredoxin system a model experiment of the superoxide anion radical production by metalloflavoproteins. Biochem Biophys Res Commun 41 177-183, 1970. 

It was pointed out previously that both bacterial and plant fer-redoxins are colored proteins in the oxidized state. Fig. 3 shows the visible and ultraviolet absorption spectra of a bacterial (C. pasteurianum) and plant (spinach) ferredoxin. Bacterial ferredoxin shows a single peak in the visible region at 390 m(r and a peak in the ultraviolet region at about 280 mp. with a shoulder at 300 mp. The relative height of the peak at 280 mp to the shoulder at 300 mp varies among preparations from different bacteria generally the peak at 280 mp predominates (Loven-berg, Buchanan, and Rabinowitz (65) Bachofen and Arnon (12)). Plant... 

Apella and San Pietro (4) Fry and San Pietro (47). Arnon (5) estimates spinach ferredoxin to have a molecular weight of 13,000 on the basis of iron content. 

The values for the molecular weight of plant ferredoxins reported by different investigators are not in agreement. Appella and San Pietro 4) reported a molecular weight of 17,000 for spinach ferredoxin, based on an S2o,w of 1.36 and a diffusion coefficient of 6.6 X 10 7 cm2/sec. The partial specific volume was assumed to be 0.71. A calculation by the writer for the minimal molecular weight of spinach ferredoxin from available amino acid composition data Fry and San Pietro (47)) and the assumption of six cysteine residues per mole gave a value of 13,000. Arnon (5)... 

In an elegant experiment coupling the oxidation of reduced spinach ferredoxin to the reduction of TPN (a two electron carrier) by a crystalline enzyme, Whatley, Tagawa, and Arnon (114) showed that, like the cytochromes, the reduction of spinach ferredoxin involves the transfer of a single electron. These results were confirmed by Fry et al- (45) for spinach ferredoxin. Sobel and Lovenbarg (96) applied this same technique, as well as hydrogen-hydrogenase, to C. pasteurianum ferredoxin and found that its oxidation and reduction involved transfer of two, rather than one, electron. Reduction of this bacterial ferredoxin was accompanied by the reduction of two ferric iron atoms to the ferrous state. 

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