Dithionite-reduced proteins, Mossbauer

Mossbauer spectroscopy of AvF clearly demonstrated the presence of P clusters (174). The EPR spectra of dithionite-reduced VFe proteins are complex, indicating the presence of several paramagnetic species. Avl exhibits broad EPR signals, with g values of 5.8 and 5.4 integrating to 0.9 spins per V atom, which have been assigned to transitions from the ground and first excited state of a spin S = system (175). EPR data for AcF are more complex, with g values at 5.6, 4.3, and 3.77 that appear to arise from a mixture of S = species (176). The signals were associated with a midpoint potential of... 

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. 

Mossbauer spectroscopy of Fe-enriched MoFe protein in dithionite-reduced and dye-oxidized oxidation states were interpreted in terms of approximately 50% of the Fe in the protein being present in cubane clusters similar to [4Fe-4S] clusters of simpler Fe-S proteins, e.g., ferredoxins and Chromatium high-potential iron proteins. Spectra of MoFe protein in which P clusters were selectively enriched with Fe were consistent with two of the clusters having slightly different environments. 

Progress in the biochemistry and spectroscopy of iron-only nitrogenase has occurred, with the most active, pure preparations reported in 1997. Mossbauer and EXAFS results support electronic and structural analogy between the eight-iron cluster in this enzyme (the FeFeco ) and the FeMoco. One notable difference is that the FeFeco is diamagnetic in its dithionite-reduced form, and thus corresponds to M° in the MoFe protein. Therefore, the oxidation states are thought to be Fe(II)4Fe(III)4 or Fe(II)6Fe(III)2, in analogy to the two most likely oxidation... 

Attempts to study the nature of Fe in the MoFe protein have extended to oxidation studies on Avl and Kpl. From Mossbauer studies Orme-Johnson et al. (1977) defined four states of Fe in native dithionite-reduced Avl. These included (8 Fe/mol, Mg in Kpl) state D (9 Fe/mol equivalent to the epr silent 8-Fe group Mg in Kpl) Fe (3 Fe/mol equivalent to the 2-Fe group M4 in Kpl) and state S (1 Fe/mol) which may be an impurity (Munck et al., 1975). In a later study Zimmerman et al. (1978) modified these numbers they concluded that M, ccmtained 12 Fe in two apparently identical clusters. Species D and Fe " were replaced by four P clusters each containing three irons of the D type and one of the Fe " type. The remaining irons were in species S (2/mol) which, although inert under all conditions tested, were present in Cpl, Avl, and Kpl. Presumably, species S is not an impurity. The authors emphasized that active MoFe protein should contain 30 2 Fe/mol and that the P clusters, although spectroscopically unique, were probably closely related to conventional 4Fe4S clusters. 

An interesting fact is that when Fdll is converted into the apoprotein by treatment with trichloroacetic acid and the iron-sulfur center is reconstituted by the addition of iron and sulfide171 the protein becomes a [4 Fe-4 S] ferredoxin. Indeed the isotropic type signal of the oxidized form is no longer observed and upon reduction with dithionite a g = 1.94 type signal indicative of a + 1 oxidation state is observed at 4.2 K (see Fig. 8). This reconstituted material exhibits Mossbauer spectra almost identical to the observed for the reduced ferredoxin from Bacillus stearother-mophilus46) a protein for which a [4 Fe-4 S] center is firmly established (Fig. 9). 

The evidence in the foregoing sections indicates that the Fe protein accepts electrons from the electron donor and that the MoFe protein binds the reducible substrate. Evidence from several sources and techniques, principally epr and Mossbauer spectroscopy and stopped-flow spectrophotometry, shows that electrons pass from the Fe protein to the MoFe protein with the concomitant hydrolysis of ATP. The earliest evidence came from steady-state epr studies on the nitrogenase of K. pneumoniae (Smith et al., 1972), A. vinelandii and C. pasteurianum (Orme-Johnson et al., 1972 Palmer et al., 1972 Zumft et al., 1972). In the presence of sodium dithionite and without MgATP, the epr spectra of the two proteins are additive. When ATP is added both spectra are largely bleached within (it is now agreed) the turnover time (185 ms/electron pair at 23 C) of the enzyme and remain so imtil the dithio-... 

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