The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed.

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Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.

Subtilisin E was rationally engineered to improve its stability in polar organic solvents such as dimethylformamide (DMF). A charged surface residue, Asp248, was substituted by three amino acids of increasing hydrophobicity, Asn, Ala, and Leu; all three variants were stabilized with respect to wild type in 80% DMF. This stabilization was only observed in the presence of high concentrations of the organic solvent: no stability enhancements were observed in 40% DMF. In contrast, the mutation Asn218 --> Ser alters internal hydrogen bonding interactions and stabilizes subtilisin E in both 40% and 80% DMF. This study provides additional evidence that substitution of surface-charged residues is a generally useful mechanism for stabilizing enzymes in organic media and that the stabilizing effects of such substitutions are unique to highly altered solvent environments. The effects of the single amino acid substitutions on free energies of stabilization are additive in the Asp248 --> Asn + Asn218 --> Ser combination variant, yielding an enzyme that is 3.4 times more stable than wild type in 80% DMF.

Strains of Azotobacter vinelandi which contain defined deletions within the nijD and nijK genes which encode, respectively, the a and 13 subunits of the MoFe protein of nitrogenase were analyzed. When synthesized without its partner, the 1 subunit accumulated as a soluble 14 tetramer. In contrast, when the a subunit was present without its partner, it accumulated primarily as an insoluble aggregate. The solubility of this protein was increased by the presence of a form of the 13 subunit which contained a large internal deletion, such that the a subunit could participate in the assembly of small amounts of an a212 holoprotein. When synthesized alone, the 13 subunit was remarkably stable, even when the protein contained a large internal deletion. The a subunit, however, was much more rapidly degraded than the 13 subunit, both when it was synthesized alone in its native background and when it was synthesized with its 1 subunit partner in a foreign background. Antibodies raised against purified a2132 MoFe protein recognized epitopes only on the nondenatured 1 subunit and not on the nondenatured a subunit. Our findings that all epitopes for the a2P2 tetramer appeared to be on the 1 subunit, that the 1 subunit assembled-into P4 tetramers, and that the a subunit alone was very insoluble, combined with the previous finding that the Fe protein binds to the 1 subunit (A. H. Willing, M. M. Georgiadis, D. C. Rees, and J. B. Howard, J. Biol. Chem. 264:849948503, 1989) all suggest that the 13 subunit has a more surface location than the a subunit in the a212 tetramer.Nitrogenase catalyzes the biological reduction of N2 to NH3. Molybdenum nitrogenases from diverse procaryotes are composed of two separately purified proteins called the iron protein (Fe protein), and the molybdenum-iron protein (MoFe protein), both of which are required for substrate reduction (28,34,36). The Fe protein from one organism can complex with the MoFe protein from another to yield an active enzyme (13, 14).The MoFe proteins are a232 tetramers with molecular weights of 210,000 to 250,000. The a and 1 subunits encoded by the nijfl and ni/K genes, respectively, are similar in size (29,40). The arrangement of the subunits is unknown, but information from neutron small-angle scattering (26), electron microscopy (35, 39), and X-ray diffraction studies (43) points toward an aspherical, probably pseudotetrahedral particle with twofold molecular symmetry. Current models suggest six metal centers within the a2532 tetramer (25,28,34,36), two of which are FeMo cofactor clusters. The FeMo cofactor, at the site of substrate binding and reduction (18), contains about six iron atoms, eight sulfur atoms, and one molybdenum atom arranged in a novel spin-coupled cluster (28,34,36) with one molecule of homocitrate (19). The other four metal centers appear to be [4Fe-4S]-type clusters in an unusual (zero) oxidation state (22, 25) with some noncysteine ligation (11,22,25,40); they are not all identical and appear to occupy two different environments (25).The relationship...

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