Role for the nitrogenase MoFe protein α-subunit in FeMo-cofactor binding and catalysis

Scott, Deborah J.; May, Harold D.; Newton, William E.; Brigle, Kevin E.; Dean, Dennis R.

doi:10.1038/343188a0

Cited by 136 publications

(106 citation statements)

References 30 publications

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“…These cytochromes are found in most eukaryotes and in a range of prokaryotes (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). They are oxidative enzymes involved in detoxification.…”

Section: Discussionmentioning

confidence: 99%

“…The reduction of acetylene by strains CA115 and CA116 coupled with their inability to fix N 2 indicates that the anfO and anfR products are required for the reduction of N 2 but not for acetylene reduction. Previous studies show that the reduction of N 2 to NH 3 requires more specific cofactor-polypeptide interactions than does the reduction of other substrates (8,29,34,35). Thus, the anfO and anfR products could be involved in cofactor-polypeptide interactions in dinitrogenase 3.…”

Section: N/mentioning

confidence: 99%

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Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

et al. 1996

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In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo-and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15 N 2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo-and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative -independent transcription termination site has been identified 250 bp from the 3 end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo-and V-deficient conditions.Nitrogen fixation, the reduction of dinitrogen to ammonia, is an enzymatic process that occurs in a wide range of bacterial species, including the soil bacterium Azotobacter vinelandii. This process is catalyzed by the enzyme complex nitrogenase. Nitrogenase is a metalloenzyme composed of two protein components called dinitrogenase reductase and dinitrogenase. A. vinelandii harbors three genetically distinct nitrogenases (3, 4). The first nitrogenase is the molybdenum (Mo)-containing enzyme (nitrogenase 1) that is made in the absence of a fixed nitrogen source when Mo is available (9). The second enzyme, a vanadium (V)-containing nitrogenase (nitrogenase 2), is synthesized under Mo-deficient conditions in the presence of V (4, 9, 12). The third nitrogenase (nitrogenase 3) is expressed only in the absence of Mo and V (3,6,17,30).The protein subunits for the Mo nitrogenase are encoded by the nifHDK operon (10). The structural genes for V nitrogenase comprise two operons, vnfHorfFd and vnfDGK (12). The subunits for the third nitrogenase are encoded by the anfHDGK operon (11).Recently, three other diazotrophs, Clostridium pasteurianum (41), Rhodobacter capsulatus (16, 33), and Rhodospirillum rubrum (4), were found to have alternative nitrogenases that are similar to nitrogenase 3 of A. vinelandii. These nitrogenases are expressed only in the absence of Mo. Nitrogenase 3 is composed of dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase reductase 3 is a homodimer (␥ 2 ) encoded by the anfH gene, while dinitrogenase 3 is a hexamer (␣ 2 ␤ 2 ␦ 2 ). The ␣-and ␤-subunits are encoded by the anfD and anfK genes, respectively. The ␦-subunit, a small protein, characteristic of the alternative nitrogenases (33,39,41), is encoded by the anfG gene.In addition to the struct...

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“…These cytochromes are found in most eukaryotes and in a range of prokaryotes (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). They are oxidative enzymes involved in detoxification.…”

Section: Discussionmentioning

confidence: 99%

Section: N/mentioning

confidence: 99%

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

et al. 1996

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“…Not only did such primary sequence identities give credence to the scaffold hypothesis (i.e., the nifEN gene products form a complex upon which FeMo-co is preassembled), but they also permitted the correct prediction of some, but not all, the FeMo-co-binding domains located within the MoFe protein (5). Moreover, the identification of such domains provided, prior to the determination of the crystal structures, a rational strategy for development of amino acid substitution studies aimed at determining the contribution of the FeMo-co polypeptide environment to catalysis (46).…”

Section: Intermolecular Electron Transfermentioning

confidence: 99%

Nitrogenase metalloclusters: structures, organization, and synthesis

1993

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“…The ability of CA11.1.79 to reduce acetylene coupled with its inability to grow diazotrophically in Mo-deficient media suggests that the anfG product is required for the reduction of N 2 but not for acetylene reduction. There is evidence that the reduction of N 2 to NH 3 requires more specific cofactorpolypeptide interactions than the reduction of other substrates (9,36,39). Thus, the anfG product may be involved with cofactor-polypeptide interactions in dinitrogenase 3 (31).…”

Section: Vol 177 1995mentioning

confidence: 99%

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

et al. 1995

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vnfG and anfG encode the ␦ subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf ؊ Anf ؊ strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the ␦ subunits are not required for acetylene reduction by nitrogenases 2 and 3.

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Role for the nitrogenase MoFe protein α-subunit in FeMo-cofactor binding and catalysis

Cited by 136 publications

References 30 publications

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

Nitrogenase metalloclusters: structures, organization, and synthesis

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

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