Iron-sulfur proteins, Mossbauer

The relevant structural and chemical information on iron-sulfur proteins provided by EPR, ENDOR and Mossbauer techniques serves as an instructive example for the success of combining different types of spectroscopic methods. 

One large class of non-heme iron-containing biomolecules involves proteins and enzymes containing iron-sulfur clusters. Iron-sulfur clusters are described in Sections 1.7 (Bioorganometallic Chemistry) and 1.8 (Electron Transfer) as well as in Section 3.6 (Mossbauer Spectroscopy). See especially Table 3.2 and the descriptive examples discussed in Section 3.6.4. Iron-sulfur proteins include rubredoxins, ferrodoxins, and the enzymes aconitase and nitrogenase. The nitrogenase enzyme was the subject of Chapter 6 in the hrst edition of this text—see especially Section 6.3 for a discussion of iron-sulfur clusters. In this... 

This article will limit its view to hemoproteins and heme prosthetic groups, the iron-sulfur proteins, and hemerythrin. Mossbauer spectro-... 

Iron atoms in states other than Fe(II) and Fe(III) are rare in biological material, but there is one case where Mossbauer evidence has pointed to an Fe(IV) electronic configuration. Horseradish peroxidase, when it forms peroxide derivatives (Compounds I and II of HRP), displays an isomer shift which is about equal to that obtained with Fe metal (23). A similar observation has also been found on an analogous compound, Japanese Radish Peroxidase (72). There is no evidence for Fe(I) or Fe (IV) states in any other hemoproteins, or in any of the iron-sulfur proteins. 

The second class of iron-containing proteins which have been well-studied by Mossbauer spectroscopy, and by other resonance techniques, are the iron-sulfur proteins. These molecules are also known by the name, ferredoxins. Iron-sulfur proteins in several varieties serve as electron-transport agents for processes in plants, bacteria, and mammals. Perhaps the most-studied physiological process involving the iron-sulfur proteins is the study of their role in photosynthesis. This subject has been extensively reviewed by Arnon 126,135), Hind and Olson 127), Hall and... 

Table 1 lists some of the properties of the plant-type iron sulfur-proteins for which extensive study by EPR and Mossbauer spectroscopy has been reported. The physical properties summarized show that the plant-type iron sulfur proteins have molecular weights in the range from 12,000 to 24,000 and have EPR g-values (gx, gy, gz) all of the g = 1.94" type shown in Fig. 6 but with minor variations reflecting axial or nonaxial symmetry of the paramagnetic center. The amino-acid sequences of four plant-type iron-sulfur-proteins are known alfalfa (136), L. glauca (137), Scenedesmus (138), and spinach (139). Each protein has about 97 residues, all in a single peptide chain these are shown in Table 2. 

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...

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 first Mossbauer spectroscopic studies on this class of iron-sulfur proteins were carried out by Blomstrom et al. 174) who reported only spectra taken on the oxidized form of the protein from Clostridium pasteuranium. Evidence in the form of two partially resolved quadrupole pairs was presented to support two distinct environments for the iron in the oxidized form of the protein, the ratio of 5 2 was suggested in keeping with the then thought number of Fe atoms per protein molecule. This assignment of the number of Fe atom per site, of course, rests on the assumption of equal Lamb-Mossbauer recoil-free fractions for the two sites. 

The report-in-depth on Mossbauer spectroscopy of the iron-sulfur proteins includes collaborative research, also published elsewhere, with W. H. Onne-Johnson, Graham Palmer, R. H. Sands, and I. Salmeen. 

This cluster formally contains three iron(III) and one iron(E). It is present in a class of proteins called high potential iron-sulfur proteins (HiPIP). It has also been prepared through oxidation of [(RS)4Fe4S4]2 model compounds [57]. Both in the model compound at low temperatures and in proteins there is electron delocalization on one mixed valence pair [58-62]. Therefore, the polymetallic center is constituted by two iron ions at the oxidation state +2.5 and two iron ions at the oxidation state +3. Hamiltonian (6.20), or a more complicated one [40, 41,43], can be used to describe the electronic structure. Indeed, a delocalization operator is sometimes needed in the Hamiltonian [40,41,43]. Consistently with magnetic Mossbauer data the S M subspin involving the mixed valence pair is 9/2, whereas the S n subspin involving the iron(IH) ions is 4. Mossbauer and EPR data do not exclude % and 3, respectively, for the two pairs [57] in any case, the... 

EPR and Mossbauer study of two mutants of the mononuclear iron sulfur protein rubredoxin72 compared the results of chemical and cryoradiolytic reduction and concluded that the latter reduction method produces exclusively one reduced species (serine coordinated iron known from the X-ray structure of the ferric protein) because it leaves the coordination of the ferric precursor unaltered. The identity of another spectral form observed in the chemically reduced C42S mutant in solution could not be unambiguously identified. 

Cammack, R., Dickson, D. P. E., Johnson, C. E. Evidence from Mossbauer spectroscopy and magnetic resonance on the active centers of the iron-sulfur proteins, in Ref. 12, p. 283... 

Detailed studies on and Fb have been hampered by the property that their EPR spectra are not additive. This property has been attributed to a magnetic interaction between reduced F and Fg, indicating that they are very close to each other. The values of F and Fg are -540 and -590 mV respectively in spinach PS I particles. Their values are always in that range, but their relative values vary in different plant species for example, F has a more negative than Fb in barley and in a halophilic alga. The shape and temperature dependence of the EPR spectra of F and Fb are typical of iron-sulfur proteins. They are considered to be 4Fe-4S centers, since after modification by dimethyl sulfoxide their spectrum is characteristic of 4Fe-4S centres and because their Mossbauer spectra are also in agreement with that attribution. The presence of 10-12 Fe-S pairs in each PS I centre is compatible with this assignment (for reviews, see Refs. 25 and 26). 

A third species, Fx, the spectrum of which considerably deviates from that of a ferredoxin, is observed under highly reducing conditions [43,44], From Mossbauer studies it was calculated that Fx is a 4Fe-4S iron-sulfur protein [45], It is still not quite certain, however, whether under physiological conditions Fx really acts as an obligatory electron acceptor. In spite of the above-mentioned uncertainties, EPR is the only technique that is capable of furnishing detailed information on the various iron-sulfur protein acceptors their optical absorbance difference spectra all show a rather uninformative weak band around 430 nm,... 

The fully characterization of both simple or more complex (but well defined in terms of active center composition) iron-sulfur proteins leads to a well of information. The compilation of typical spectroscopic features of the known basic iron-sulfur structures enables a preliminary characterization of centers in a new simple situation or even in some more complex ones. EPR spectroscopy of the iron-sulfur cores in the appropriated oxidation states have characteristics that can be used to readily distinguish certain type of centers58). This technique has also been used to analyse components in complex systems. However the use of EPR as the sole technique can be misleading, when applied to new situations as we have seen for the case of the [3 Fe—xS] core. In this particular case only the conjunction of EPR and Mossbauer can lead to a proper characterization27 33 34 55). 

Debrunner, P.G., et aL Recent Mossbauer Results of some Iron-Sulfur Proteins and Model Complexes. In Iron-Sulfur Proteins (ed. Lovenberg, W.), Vol. 3, New York, Academic Press 1977, pp. 381-417... 

Miinck, E., Huynh, B. H. Iron-Sulfur Proteins Combined Mossbauer and EPR Studies. 

Ragsdale SW, Lindahl PA, Miinck E (1987) Mossbauer, EPR, and optical studies of the corrinoid/ iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum. J Biol Chem 262 14289-14297... 

Before leaving iron-sulfur proteins, we must mention the important contributions that model studies have made, in particular before protein X-ray structural data were available. For discrete clusters of the t5 pe formed by reaction 28.15 and shown in diagram 28.20, it is possible to investigate magnetic, electronic spectroscopic and electrochemical properties, record Fe Mossbauer spectra (see Section 2.12) and determine accurate structural data by X-ray diffraction. Working with metalloproteins is, of course, far more difficult. 

A number of chemical structures for Fd have been suggested. The chelate structure [Fig.(l)] is based on chemical analysis, ESR and Mossbauer spectroscopy (see also Iron-sulfur proteins). Model experiments led to the construction of metal-atom island structures, or clusters. In this model, the labile sulfur... 

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