PubMed Health⌕ Search

Biomedical subjects

Markus W Ribbe

Publications and source records attributed to Markus W Ribbe.

15 recordsLinked to original sources
PubMed2007

Molecular insights into nitrogenase FeMo cofactor insertion: the role of His 362 of the MoFe protein alpha subunit in FeMo cofactor incorporation.

The assembly of the complex iron-molybdenum cofactor (FeMoco) of nitrogenase molybdenum-iron (MoFe) protein has served as one of the central topics in the field of bioinorganic chemistry for decades. Here we examine the role of a MoFe protein residue (His alpha362) in FeMoco insertion, the final step of FeMoco biosynthesis where FeMoco is incorporated into its binding site in the MoFe protein. Our data from combined metal, activity and electron paramagnetic resonance analyses show that mutations of His alpha362 to small uncharged Ala or negatively charged Asp result in significantly reduced FeMoco accumulation in MoFe protein, indicating that His alpha362 plays a key role in the process of FeMoco insertion. Given the strategic location of His alpha362 at the entry point of the FeMoco insertion funnel, this residue may serve as one of the initial docking points for FeMoco insertion through transient ligand coordination and/or electrostatic interaction.

Azotobacter vinelandii↗
PubMed2006

Variable-temperature, variable-field magnetic circular dichroism spectroscopic study of the metal clusters in the DeltanifB and DeltanifH mofe proteins of nitrogenase from Azotobacter vinelandii.

Deletion of nifB results in the formation of a variant nitrogenase MoFe protein (DeltanifB MoFe protein) that appears to contain two normal [8Fe-7S] P clusters. This protein can be reactivated to form the holo MoFe protein upon addition of isolated FeMo cofactor. In contrast, deletion of nifH results in a variant protein (DeltanifH MoFe protein) that appears to contain FeS clusters different from the normal P cluster, presumably representing precursors of the normal P cluster. The DeltanifH MoFe protein is not reconstituted to the holo MoFe protein with isolated FeMo cofactor. The EPR and EXAFS spectroscopic properties of FeS clusters in the DeltanifH MoFe protein clearly differ from those of the normal P cluster found in the DeltanifB MoFe protein and suggest the presence of [4Fe-4S]-like clusters. To further characterize the metal cluster structures in the DeltanifH MoFe protein, a variable-temperature, variable-field magnetic circular dichroism (VTVH-MCD) spectroscopic study has been undertaken on both the DeltanifB MoFe protein and the DeltanifH MoFe protein in both the dithionite-reduced and oxidized states. This study clearly shows that each half of the dithionite-reduced DeltanifH MoFe protein contains a [4Fe-4S]+ cluster paired with a diamagnetic [4Fe-4S]-like cluster. Upon oxidation, the VTVH-MCD spectrum of the DeltanifH MoFe protein reveals a paramagnetic, albeit EPR-silent system, suggesting an integer spin state. These results suggest that the DeltanifH MoFe protein contains a pair of neighboring, unusual [4Fe-4S]-like clusters, which are paramagnetic in their oxidized state.

Azotobacter vinelandii↗
PubMed2006

Nitrogenase Fe protein: A molybdate/homocitrate insertase.

The Fe protein is indispensable for nitrogenase catalysis and biosynthesis. However, its function in iron-molybdenum cofactor (FeMoco) biosynthesis has not been clearly defined. Here we show that the Fe protein can act as a Mo/homocitrate insertase that mobilizes Mo/homocitrate for the maturation of FeMoco precursor on NifEN. Further, we establish that Mo/homocitrate mobilization by the Fe protein likely involves hydrolysis of MgATP and protein-protein interaction between the Fe protein and NifEN. Our findings not only clarify the role of the Fe protein in FeMoco assembly and assign another function to this multitask enzyme but also provide useful insights into a mechanism of metal trafficking required for the assembly of complex metalloproteins such as nitrogenase.

Azotobacter vinelandii↗
PubMed2006

FeMo cofactor maturation on NifEN.

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 Fe/S 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.

Electron Spin Resonance Spectroscopy↗
PubMed2006

Molecular insights into nitrogenase FeMoco insertion: TRP-444 of MoFe protein alpha-subunit locks FeMoco in its binding site.

Biosynthesis of the FeMo cofactor (FeMoco) of nitrogenase MoFe protein is arguably one of the most complex processes in metalloprotein biochemistry. Here we investigate the role of a MoFe protein residue (Trp-alpha444) in the final step of FeMoco assembly, which involves the insertion of FeMoco into its binding site. Mutations of this aromatic residue to small uncharged ones result in significantly decreased levels of FeMoco insertion/retention and drastically reduced activities of MoFe proteins, suggesting that Trp-alpha444 may lock the FeMoco tightly in its binding site through the sterically restricting effect of its bulky, aromatic side chain. Additionally, these mutations cause partial conversion of the P-cluster to a more open conformation, indicating a potential connection between FeMoco insertion and P-cluster assembly. Our results provide some of the initial molecular insights into the FeMoco insertion process and, moreover, have useful implications for the overall scheme of nitrogenase assembly.

Azotobacter vinelandii↗
PubMed2006

Structural insights into a protein-bound iron-molybdenum cofactor precursor.

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron- and sulfur-containing FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe-4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco.

Azotobacter vinelandii↗
PubMed2005

Nitrogenase reactivity with P-cluster variants.

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.

Azotobacter vinelandii↗
PubMed2005

Identification of a nitrogenase FeMo cofactor precursor on NifEN complex.

The biosynthesis of the FeMo cofactor (FeMoco) of Azotobacter vinelandii nitrogenase presumably starts with the production of its Fe/S core by NifB (the nifB gene product). This core is subsequently processed on the alpha2beta2 tetrameric NifEN complex (formed by the nifE and nifN gene products). In this article, we identify a NifEN-bound FeMoco precursor form that can be converted to fully assembled FeMoco in a so-called FeMoco-maturation assay containing only purified components. We also establish that only molybdate, homocitrate, MgATP, and Fe protein are essential for FeMoco maturation. The FeMoco-maturation assay described here will further address the remaining questions related to the assembly mechanism of the ever-intriguing FeMoco.

Azotobacter vinelandii↗
PubMed2004

Competitive 15N kinetic isotope effects of nitrogenase-catalyzed dinitrogen reduction.

Biological N2 fixation is achieved under ambient conditions by enzymatic catalysis. The enzyme nitrogenase has been studied extensively, but the N2 chemical reduction step is, by far, not rate limiting and hard to examine. A new method was developed that allows studying the reduction transition state within the enzyme's complex kinetic cascade by means of the 15N kinetic isotope effect on the reaction's second-order rate constant, V/K. A value of 1.7% +/- 0.2% was measured.

Azotobacter vinelandii↗
PubMed2004

Characterization of Azotobacter vinelandii nifZ deletion strains. Indication of stepwise MoFe protein assembly.

The nifZ gene product (NifZ) of Azotobacter vinelandii has been implicated in MoFe protein maturation. However, its exact function in this process remains largely unknown. Here, we report a detailed biochemical/biophysical characterization of His-tagged MoFe proteins purified from A. vinelandii nifZ and nifZ/nifB deletion strains DJ1182 and YM6A (Delta nifZ and Delta nifZ Delta nifB MoFe proteins, respectively). Our data from EPR, metal, activity, and stability analyses indicate that one alpha beta subunit pair of the Delta nifZ MoFe protein contains a P cluster ([8Fe-7S]) and an iron-molybdenum cofactor (FeMoco) ([Mo-7Fe-9S-X-homocitrate]), whereas the other contains a presumed P cluster precursor, possibly comprising a pair of [4Fe-4S]-like clusters, and a vacant FeMoco site. Likewise, the Delta nifZ Delta nifB MoFe protein has the same composition as the Delta nifZ MoFe protein except for the absence of FeMoco, an effect caused by the deletion of the nifB gene. These results suggest that the MoFe protein is likely assembled stepwise, i.e. one alpha beta subunit pair of the tetrameric MoFe protein is assembled prior to the other, and that NifZ might act as a chaperone in the assembly of the second alpha beta subunit pair by facilitating a conformational rearrangement that is required for the formation of the P cluster through the condensation of two [4Fe-4S]-like clusters. The possibility of NifZ exercising its effect through the Fe protein was ruled out because the Fe proteins from nifZ and nifZ/nifB deletion strains are not defective in their normal functions. However, the detailed mechanism of how NifZ carries out its exact function in MoFe protein maturation awaits further investigation.

Azotobacter vinelandii↗
PubMed2004

Comparison of iron-molybdenum cofactor-deficient nitrogenase MoFe proteins by X-ray absorption spectroscopy: implications for P-cluster biosynthesis.

Nitrogenase, the enzyme system responsible for biological nitrogen fixation, is believed to utilize two unique metalloclusters in catalysis. There is considerable interest in understanding how these metalloclusters are assembled in vivo. It has been presumed that immature iron-molybdenum cofactor-deficient nitrogenase MoFe proteins contain the P-cluster, although no biosynthetic pathway for the assembly of this complex cluster has been identified as yet. Through the comparison by iron K-edge x-ray absorption edge and extended fine structure analyses of cofactor-deficient MoFe proteins resulting from nifH and nifB deletion strains of Azotobacter vinelandii, a novel [Fe-S] cluster is identified in the DeltanifH MoFe protein. The iron-iron scattering displayed by the DeltanifH MoFe protein is more similar to that of a standard [Fe(4)S(4)]-containing protein than that of the DeltanifB MoFe protein, which is shown to contain a "normal" P-cluster. The iron-sulfur scattering of the DeltanifH MoFe protein, however, indicates differences in its cluster from an [Fe(4)S(4)](Cys)(4) site that may be consistent with the presence of either oxygenic or nitrogenic ligation. Based on these results, models for the [Fe-S] center in the DeltanifH MoFe protein are constructed, the most likely of which consist of two separate [Fe(4)S(4)] sites, each with some non-cysteinyl coordination. This type of model suggests that the P-cluster is formed by the condensation of two [Fe(4)S(4)] fragments, possibly concomitant with Fe protein (NifH)-induced conformational change.

Azotobacter vinelandii↗
PubMed2002

Direct assessment of the reduction potential of the [4Fe-4S](1+/0) couple of the Fe protein from Azotobacter vinelandii.

Recently, it has been demonstrated that the [4Fe-4S] cluster of the Fe protein of nitrogenase from Azotobacter vinelandii can be reduced to an unprecedented all-ferrous state. In this work, the reduction potential for the formation of the all-ferrous state is measured by the reactions of the reduced and oxidized Fe protein with a variety of chemical redox active agents, and by mediated spectroelectrochemical titration. Redox titrations obtain a potential ca. -790 mV/NHE for the formation of the all-ferrous state, a value consistent with the chemical reactivity experiments and with recent theoretical calculations. At present, no known redox protein in A. vinelandii is capable of generating the all-ferrous Fe protein.

Azotobacter vinelandii↗
PubMed2002

The FeMoco-deficient MoFe protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster features.

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (Delta(nifH) MoFe protein) was purified in large quantity. The alpha(2)beta(2) tetrameric Delta(nifH) MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The Delta(nifH) MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g approximately 2 region. The indigo disulfonate-oxidized Delta(nifH) MoFe protein does not show features of the P(2+) state of the P-cluster of the Delta(nifB) MoFe protein. The oxidized Delta(nifH) MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S](1+) cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S](1+) cluster of the Fe protein. Furthermore, the dithionite-reduced Delta(nifH) MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.

Azotobacter vinelandii↗
PubMed2002

Structure of a cofactor-deficient nitrogenase MoFe protein.

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits. In this structure, one of the three alpha subunit domains exhibits a substantially changed conformation, whereas the rest of the protein remains essentially unchanged. A predominantly positively charged funnel is revealed; this funnel is of sufficient size to accommodate insertion of the negatively charged cofactor.

Amino Acid Sequence↗