Molybdenum Determination of Soils and Rocks with Dithiol

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Rebelein

et al. 2016

NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster-bound NifEN (designated NifEN M ) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifEN M upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN M with an M-cluster in one αβ-half and an empty clusterbinding site in the other αβ-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αβ-dimers of NifEN. Perhaps most importantly, NifEN M displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αβ-dimer of NifEN M as a structural and functional mimic of one catalytic αβ-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase.nitrogenase | catalysis | assembly | functional homolog | NifEN N itrogenase plays a key role in global nitrogen cycle through its unique ability to reduce nitrogen to ammonia under ambient conditions. The best characterized, Mo-dependent nitrogenase uses a two-component system for substrate reduction, in which a specific reductase (designated the Fe protein) serves as the obligate electron donor for a catalytic component (designated the MoFe protein) during catalysis (1). The Fe protein (or NifH) is a γ 2 -dimer, which contains a [Fe 4 S 4 ] cluster between the two subunits and an ATP-binding site within each subunit, whereas the MoFe protein (or NifDK) is an α 2 β 2 -tetramer, which contains a P-cluster ([Fe 8 S 7 ]) between each α/β-subunit interface and an M-cluster (also termed the cofactor; [MoFe 7 S 9 C-homocitrate]) within each α-subunit (2-4). Catalysis of nitrogenase involves formation of a functional complex between the two component proteins of the Mo-nitrogenase (5), which permits ATP-dependent transfer of electrons from the [Fe 4 S 4 ] cluster of NifH, via the P-cluster, to the M-cluster of NifDK and the subsequent reduction of substrate at the M-cluster site upon accumulation of a sufficient amount of electrons.Arguably the most complex metallocluster ...

Section: Methodsmentioning

confidence: 99%

Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component

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Rebelein

et al. 2016

“…Cell growth, protein purification, preparation of apo Fe protein, FeMoco maturation assay, metal analysis, visible region absorption, EPR and x-ray spectroscopies were performed as described (13,17,18,24,33,34 AMPPNP . These were control assays with the same conditions as i except that ATP was replaced by 2.4 mM ADP, adenosine 5Ј-O-(3-thiotriphosphate) (ATP␥S) or 5Ј-adenylylimidodiphosphate (AMPPNP).…”

Section: Methodsmentioning

confidence: 99%

FeMo cofactor maturation on NifEN

Corbett

et al. 2006

163

FeMo cofactor (FeMoco) biosynthesis is one of the most complicated processes in metalloprotein biochemistry. Here we show that Mo and homocitrate are incorporated into the Fe͞S core of the FeMoco precursor while it is bound to NifEN and that the resulting fully complemented, FeMoco-like cluster is transformed into a mature FeMoco upon transfer from NifEN to MoFe protein through direct protein-protein interaction. Our findings not only clarify the process of FeMoco maturation, but also provide useful insights into the other facets of nitrogenase chemistry. (9), which is located at the ␣␤-interface and ligated to six protein residues; and the [Mo-7Fe-9S-X-homocitrate] (the identity of X is unknown but is considered to be C, O, or N; ref. 10) FeMo cofactor (FeMoco), which is situated within the ␣-subunit and bound to only two protein residues and an exogenous homocitrate ligand. Both P-cluster and FeMoco are composed of smaller substructures: the P-cluster comprises two subclusters that share a 6 -sulfide (9) and FeMoco consists of [Mo-3Fe-3S] and [4Fe-3S] subcubanes that are bridged by three 2 -sulfides and share a central 6 -light atom (10). These metal clusters are essential for nitrogenase reaction, a process that involves ATP-dependent electron transfer from the [4Fe-

“…Ammonium was determined by a HPLC fluorescence method (37). Molybdenum (38) and iron (39) were determined as described. Vanadium was analyzed by inductively coupled plasma-MS at the chemical analysis facility of the University of Georgia (Athens).…”

Section: Methodsmentioning

confidence: 99%

Nitrogenase reactivity with P-cluster variants

Corbett

et al. 2005

Nitrogenase is a multicomponent metalloenzyme that catalyzes the conversion of atmospheric dinitrogen to ammonia. For decades, it has been generally believed that the [8Fe-7S] P-cluster of nitrogenase component 1 is indispensable for nitrogenase activity. In this study, we identified two catalytically active P-cluster variants by activity assays, metal analysis, and EPR spectroscopic studies. Further, we showed that both P-cluster variants resemble [4Fe-4S]-like centers based on x-ray absorption spectroscopic experiments. We believe that our findings challenge the dogma that the standard P-cluster is the only cluster species capable of supporting substrate reduction at the FeMo cofactor and provide important insights into the general mechanism of nitrogenase catalysis and assembly.MoFe protein ͉ VFe protein N itrogenase catalyzes one of the most remarkable chemical transformations in biological systems, the reduction of atmospheric dinitrogen to a bioavailable form, ammonia. Three classes of nitrogenase systems, namely, the Mo-, V-, and Fe-only nitrogenases, have been identified (for recent reviews see refs. 1-11). f Although encoded by different structural genes, all three nitrogenases are comprised of two essential component metalloproteins, component 1 (MoFe, VFe, or FeFe protein) and component 2 (Fe protein). g The homodimeric Fe protein of the Mo-nitrogenase has one [4Fe-4S] cluster bridged between the two subunits, whereas the ␣ 2 ␤ 2 -tetrameric MoFe protein contains two unique metal clusters per ␣␤-subunit: the [8Fe-7S] P-cluster (14), which is located at the ␣␤-interface, and the [Mo-7Fe-9S-X-homocitrate] h FeMo cofactor (FeMoco), which is situated within the ␣-subunit. ATP-dependent electron transfer is believed to proceed from the [4Fe-4S] cluster of the Fe protein to the P-cluster of the MoFe protein and finally to FeMoco where substrate reduction takes place. The Fe protein of the V-nitrogenase shares a high degree of homology with the Fe protein of the Mo-nitrogenase and, apart from the presence of an additional ␥-subunit, the VFe protein is also believed to be highly homologous to the MoFe protein with respect not only to the structural genes encoding the ␣-and ␤-subunits, but also to the redox centers contained within the protein, designated the P-cluster and the FeV cofactor (FeVco), respectively, in this case (4, 10, 16). The structurally homologous heterometal centers, i.e., FeMoco and FeVco, are also categorically termed cofactors.It is generally believed that the structural integrity of the P-cluster is indispensable for nitrogenase reactivity. However, the exact mechanism by which the P-cluster carries out its function in substrate reduction and, in particular, the oxidation states and structural conformations of the P-cluster involved in this process, remain largely a puzzle (17). Two types of cofactordeficient MoFe or VFe protein variants, generated by nifH or nifB deletion, i respectively, have been used to study the features of P-clusters without the interference of the cofactor centers...