Comparative In‐Vivo and In‐Vitro⁹⁹Mo‐Time‐Differential‐Perturbed‐Angular‐Correlation Studies on the Nitrogenase Mofe Protein and on Other Mo Species of Different N₂‐Fixing Bacteria

Abstract

Klebsiella pneumoniae, Azotobacter vinelandii and Rhodobacter capsulatus were cultivated in media containing "MOO$-. The distribution of "Mo in cells grown under conditions of repression and derepression of nitrogenase synthesis, was investigated by anion-exchange (DEAE-Sephacel) chromatography, Cells of K. pneumoniae took up MOO:-only under conditions of derepression of nitrogenase thus serving the formation of the FeMo cofactor of the MoFe protein (Kpl) as the predominant Mo-containing species. In the case of A. vinelandii, under diazotrophic growth conditions, molybdenum was preferably incorporated into the nitrogenase MoFe protein (Avl). However, if excess amounts of molybdate were present in the medium, molybdenum was also bound to the Mo-storage protein. In the presence of 20 mM NH,', conditions which completely repress nitrogenase formation, molybdenum accumulated in the Mo-storage protein exclusively. This protein proved to be unstable towards DEAE-Sephacel, apparently releasing all the molybdenum in form of MOO:-during the fractionation procedure. R. capsulatus contained, in addition to the MoFe protein (Rcl), significant amounts of other not-yet-identified Mo species, which partially are formed under conditions of both, repression and derepression of nitrogenase.The Mo centers of all these compounds were characterized by measuring the nuclear quadrupole interaction of the process "Mo(P-)"Tc using time differential perturbed angular correlation spectroscopy. Keywords: nitrogenase ; molybdenum-iron protein ; molybdenum-storage protein; time differential perturbed angular correlation (TDPAC) spectroscopy.The biological nitrogen fixation is one of the most important processes in nature, because it is essential for the N-cycle and thus for the life of all organisms (for reviews see [l-31). The conventional nitrogenase enzyme system responsible for the reduction of molecular N, to NHf consists of two separable proteins, the MoFe protein (component 1) and the Fe protein (com- whose primary role is the mediation of electrons in nitrogenase, and (b) the iron-molybdenum cofactor (FeMo cofactor, FeMoco), which is located in the a-subunits and has been postulated to be the active site for substrate (e.g. N,) reduction. Based on X-ray structure analyses of MoFe proteins from Azotobacter vinelandii and Clostridium pasteurianum, a structural cofactor model has recently been published [6, 71. According to this model the FeMo cofactor consists of two cubane fragments (Fe,S, and MoFe,S,) bridged by three inorganic sulfide ligands. The Mo center is ligated by two 0 atoms from homocitrate and one N atom from histidine of the protein side chain. In this work we determined the nuclear quadrupole interacprotein and in other Mo-containing proteins present in different (TDPAC) spectroscopy Of y-rays. This method detects the hyperfine interaction between the nuclear quadruPole moment of the probe nucleus (in our case 9 9 M~) and an electric field gradient (EFG). Since the EFG depends on the distribution tion (NQI) parameters of...

A New Type of Metalloprotein: The Mo Storage Protein from Azotobacter vinelandii Contains a Polynuclear Molybdenum–Oxide Cluster

“…In A. vinelandii , an aerobic bacterium that is widespread in soils and waters, another Mo protein exists, designated as the “molybdenum storage protein” (MoSto), which is functionally connected to nitrogen fixation3 and enables the accumulation of enormous amounts of Mo inside the cell,3–5 thus supplying the conventional nitrogenase system with Mo even in an Mo‐deficient environment. The bioavailability of Mo in soils is highly variable and may easily become a growth‐limiting factor for nitrogen‐fixing bacteria.…”

“…The only attempt ever to obtain information about the nature of the protein‐bound Mo was based on time‐differential perturbed angular correlation of γ rays (TDPAC) measurements with 99 Mo described by Müller et al5 Despite the limitations of this method (yielding a fingerprint only, no explicit structural information like interatomic distances, bond angles, etc.) and the fact that the experiments were done only with whole cells and cell‐free extracts, and not with purified protein, the TDPAC spectra gave the first hint that the Mo component in MoSto might be a polynuclear metal–oxygen cluster 5. The iron storage proteins ferritin7 and frataxin8, 9 are currently the only reported examples of biological metal storage in the form of such a cluster.…”

Exploring the Molecular Growth of Two Gigantic Half‐Closed Polyoxometalate Clusters {Mo180} and {Mo130Ce6}

“…Meanwhile, it is important to understand the way in which the additional fragments initialize the molecular growth process. This is particularly important to improve the understanding of the initial nucleation of POM clusters in solution, as well as the metal‐centered assemblies found in biological systems 5, 6. In this context, the investigation of clusters that can be grown/assembled by molecular growth processes is warranted.…”

Electroanalytical Determination of Tungsten and Molybdenum in Proteins

“…This electroanalytical determination can be an important tool to isolate new molybdenum and tungsten proteins, e.g., transport or storage proteins. Similar screenings have been performed for Clostridium acidiurici and Clostridium cylindrosporum using the radioactive isotope 185 W (28) and for Clostridium pasterianum (29,30), Klebsiella pneumoniae, Azotobacter vinelandii (31), and Rhodobacter capsulatus using 99 Mo (32). A molybdenum uptake investigation of Escherichia coli using 99 Mo has been reported (33).…”