Spectroscopy MoFe proteins

During oxidation of the MoFe protein the P clusters are the first to be oxidized at about -340 mV. This redox potential was first measured (40) using Mossbauer spectroscopy and exhibited a Nemst curve consistent with a two-electron oxidation process. It is possibly low enough for this redox process to be involved in enzyme turnover (see Section V). No additional EPR signal was observed from this oxidized form at this time. However, later a weak signal near g = 12 was detected and was finally confirmed, using parallel mode EPR... 

Pierik, A.J., Wassink, H., Haaker, H., and Hagen, W.R. 1993. Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii MoFe protein. European Journal of Biochemistry 212 51-61. 

One type of the constituent metallocenters in the MoFe protein has the properties of a somewhat independent structural entity. This component, referred to as the FeMo cofactor (FeMo-co), was first identified by Shah and Brill (1977) as the stable metallocluster extracted from acid-denatured MoFe protein. The FeMo-co was able to fully activate a defective protein in the extracts of mutant strain UW45, a protein which subsequently was shown to contain the P clusters but not the EPR-active center. The isolated cofactor accounted for the total S = t system observed by EPR and Mdssbauer spectroscopies of the holo-MoFe protein (Rawlings et al., 1978). Elemental analysis indicated a composition of Mo Fee-8 Se-g for the cofactor, which, if there are two FeMo-co s per a2 2> accounts for all the molybdenum and approximately half the iron in active enzyme (Nelson etai, 1983). Although FeMo-co has been extensively studied [reviewed in Burgess (1990)] the structure remains enigmatic. To date, all attempts to crystallize the cofactor have failed. This is possibly due to the instability and resultant heterogeneity of the cofactor when removed from the protein. Also, there is a paucity of appropriate models for spectral comparison (see Coucouvanis, 1991, for a recent discussion). Final resolution of this elusive structure may require its determination as a component of the holoprotein. 

EXAFS spectroscopy has been used to probe the molybdenum sites in the MoFe protein and the cofactor, and the iron sites in the cofactor. The Mo EXAFS data suggest the presence of four S atoms at 2.35 A, two to three Fe atoms at 2.72 A and one to two additional S atoms at about... 

The MoFe proteins from a number of bacterial sources have been isolated. Their polypeptide structures are highly conserved and their inorganic components (2 Mo 30 Fe 32 S2 per molecule) are all very similar [3], The MoFe proteins from A. vinelandii, Clostridium pasteuranium, and Klebsiella pneumoniae are denoted by Avl, Cp 1, and Kp 1, respectively. Early Mossbauer spectroscopy demonstrated [12] that the Fe atoms were probably present as clusters, and a wide range of spectroscopic techniques have been used in attempts to understand the structures of these clusters. 

When photo-electron transfer is used with a dye, such as eosin or dibromo-fluorescein in combination with NADH (and the dithionite concentration is low, ca 4 X 10" m), no dissociation of proteins is detected. Kinetic laser spectroscopy helped Syrtsova et al. [9] to follow electron transfer from the Fe protein to the MoFe protein and it was shown that, unlike the situation with dithionite, Fe protein in the complex with MoFe protein could undergo reduction by the photodonor as efficiently as in the free state in solution and electron transfer proceeds in the complex of two proteins without dissociation. 

A variety of spectroscopic and physical techniques have been used to investigate the nature of these redox centers. EPR, Fe Mossbauer spectroscopy, and Mo and Fe X-ray absorption spectroscopy Mo, Fe, and H electron-nuclear double resonance (ENDOR) linear electric field effect and magnetic circular dichroism (MCD) have provided information about the environment of the Mo and Fe nuclei and their interaction with the unpaired spin of electrons in paramagnetic species of the MoFe proteins. 

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. 

The most detailed information on the environment of Mo in MoFe proteins has been obtained from X-ray absorption spectroscopy. Analyses of the EXAFS spectra are consistent with Fe, S, and O (or N) atoms in the coordination sphere of Mo (Table III) (33-36). 

Comparison of Environments of V in VFe Proteins and Mo in MoFe Proteins Determined prom X-Ray Absorption Spectroscopy... 

Low-temperature MCD spectroscopy has been used to characterize the metal clusters present in the VFe protein of A. vinelandii (42). The temperature dependence of the MCD transitions of dithionite-reduced protein is similar to those arising from the S = 3/2 spin system of FeMoco of the MoFe proteins. The spectra of the VFe proteins have slightly different electronic and magnetic properties but provide strong spectroscopic evidence for the presence of a V-Fe-S cluster. In this oxidation state, only small additional spectral contributions from the two other paramagnetic species (S = 1/2 signal and the species detected in the dispersion mode) are observed. 

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