Mossbauer spectroscopy MoFe protein

E. Munck, et al., Nitrogenase 8. Mossbauer and EPR spectroscopy—MoFe protein component from Azotobacter vinelandii OP,... 

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... 

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. 

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. 

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... 

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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