Azotobacter Vinelandii (Iron-Molybdenum Cofactor/Nitrogen Fixation/Nijd Gene/Protein Engineering) KEVIN E

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7066-7069, October 1987 Biochemistry Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii (iron-molybdenum cofactor/nitrogen fixation/niJD gene/protein engineering) KEVIN E. BRIGLE*, ROBERT A. SETTERQUIST*, DENNIS R. DEAN*, JOHN S. CANTWELLt, MARY C. WEISSt, AND WILLIAM E. NEWTONtt§ *Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; tWestern Regional Research Center, U.S. Department of Agriculture-Agricultural Research Service, Albany, CA 94710; and tDepartment of Biochemistry and Biophysics, University of California, Davis, CA 95616 Communicated by Martin D. Kamen, July 8, 1987 ABSTRACT A strategy has been formulated for the site- Mossbauer and electron paramagnetic resonance spectros- directed mutagenesis of the Azotobacter vinelandii nifJK genes. copies (2-6), as well as biochemical and genetic studies (10), These genes encode the a and .3 subunits of the MoFe protein have been used to demonstrate that the metal centers located of nitrogenase, respectively. Six mutant strains, which produce within the MoFe protein are likely to participate in the MoFe proteins altered in their a subunit by known single amino binding and catalytic reduction of dinitrogen. However, little acid substitutions, have been- produced. Three of these direct information is available concerning the structures, transversion mutations involve cysteine-to-serine changes (at redox properties, and functions of the individual metal residues 154, 183, and 275), two involve glutamine-to-glutamic centers within the MoFe protein. Furthermore, there is no acid changes (at residues 151 and 191), and one involves an information regarding the spatial distribution of the metal aspartic acid-to-glutamic acid change (at residue 161). All three centers among or between the four subunits of the MoFe possible phenotypic responses are observed within this group- protein. One approach toward addressing these issues is to i.e., normal, slow, and no growth in the absence of a fixed- alter specifically the polypeptide environments of the indi- nitrogen source. Two-dimensional gel electrophoresis indicates vidual clusters and to determine the spectroscopic, redox, that all mutants accumulate normal levels of the subunits of and catalytic consequences that result from such alterations. both nitrogengse component proteins. Whole-cell and crude- For example, specific amino acid substitutions at key metal- extract acetylene-reduction activities indicate substantial levels center ligation points within the MoFe protein should pro- of Fe protein activity in all strains. In contrast, MoFe protein duce altered proteins with strengthened, weakened, or elim- activities do not parallel the diazotrophic growth capability for inated metallocenter interactions. Observations of these all strains. Two strains appear to exhibit altered substrate effects could indicate appropriate cluster-binding amino acid discrimination. Such analyses should aid in the identification of residues and also provide information on the mechanistic metallocluster-binding sites and subunit-subunit interaction roles of the individual clusters. For this purpose, we isolated domains of the MoFe protein and also provide insight into the the nifHDK gene cluster from Azotobacter vinelandii and mechanistic roles of the various prosthetic groups in catalysis. determined its complete nucleotide sequence (11). Here, we describe the formulation of a strategy for the site-directed Biological nitrogen fixation is catalyzed by nitrogenase (EC mutagenesis of the genes that encode the MoFe protein 1.18.6.1), a complex metalloenzyme composed of two sepa- subunits and describe the isolation and characterization ofsix rately purifiable component proteins called the Fe protein mutant A. vinelandii strains that produce altered MoFe and the MoFe protein. The Fe protein is encoded by the nifH a-subunit proteins with known amino acid substitutions. gene and acts as a specific ATP-binding, one-electron reductant of the MoFe protein, which contains the site for substrate binding and reduction (1-3). The MoFe protein is an MATERIALS AND METHODS a2f32 protein of approximately 220,000 daltons and it contains 2 Mo atoms and approximately 32 Fe and 32 S2- atoms per Oligonucleotide-Directed Mutagenesis. Oligonucleotides molecule. The a and f3 subunits are encoded by the nifD and used for mutagenesis were synthesized on a Beckman System nifK genes, respectively. Mossbauer spectroscopy (4-6) and 1 Plus DNA synthesizer. After deprotection, the oligonucle- quantitative extrusion studies indicate that the metal atoms otides were purified by HPLC (12). Oligonucleotide-directed contained within the MoFe protein are organized into at least mutagenesis was performed by the method of Zoller and six metal-containing prosthetic groups of at least two distinct Smith (13). Template DNA used for mutagenesis was a types. Approximately 16 Fe atoms can be extruded from each 1.4-kilobase A. vinelandii niJ-specific EcoRI fragment (11) MoFe protein molecule in the form of [4Fe-4S] clusters by cloned into the EcoRI site of bacteriophage vector Ml3mpl8 treatment of the native protein with thiols in a denaturing (14). Single-lane dideoxy sequencing (15) was used to screen organic solvent (7, 8). All or most of the remaining Fe and for the desired mutations. Further DNA sequence analysis both Mo atoms constitute the two identical iron-molybde- using all four reactions confirmed the mutations and estab- num cofactors (FeMoco), and these can be isolated from the lished that there were no other mutations within 400 nucle- native MoFe protein by anaerobic acid/base treatment otides surrounding the planned mutation. For each mutant (which destroys the Fe-S clusters) followed by extraction construction, replicative-form DNA carrying a known single- with N-methylformamide (9). The mutual exclusivity ofthese base-pair transversion mutation within the nifD coding region two cluster-releasing processes indicates different binding was prepared and used for transformation of A. vinelandii modes for the two cluster types. cells. Such DNA, which carries a single transversion muta- The publication costs of this article were defrayed in part by page charge Abbreviation: FeMoco, iron-molybdenum cofactor. payment. This article must therefore be hereby marked "advertisement" §To whom reprint requests should be addressed at: Western Regional in accordance with 18 U.S.C. §1734 solely to indicate this fact. Research Center, USDA-ARS, Albany, CA 94710. 7066 Downloaded by guest on September 26, 2021 Biochemistry: Brigle et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7067 tion within the niflJ coding sequence, will hereafter be called Burk medium was used to transfer cells back to the fermentor mutation-vector DNA. and incubated as above for 2.5 hr. Derepressed cells were Isolation of Mutant Strains. Transversion mutations carried harvested as above except that the cells were washed with on mutation-vector DNA were transferred to the A. vine- chilled 0.05 M Tris (pH 8.0), then centrifuged at 10,000 x g landii chromosome in either one or two steps (Fig. 1), for 10 min and stored at -80'C until needed. depending on the resultant Nif phenotype of the mutant Two-Dimensional Gel Electrophoresis. Preparation of ex- strain. Mutations resulting in a single amino acid substitution tracts for two-dimensional gel electrophoresis was performed within the nifD-encoded polypeptide may exhibit one of three as described by Bishop et al. (18). Two-dimensional gel Nif phenotypes. Namely, such mutant strains will grow analysis was performed as described by O'Farrell (19). normally, at a reduced rate, or not at all in the absence of a Enzyme Assay. Frozen cells (=13 g) were suspended in 39 fixed-nitrogen source. Mutations that cause slow growth or ml of cold 0.05 M Tris (pH 8.0) containing 1 mM Na2S204 and that have no effect on diazotrophic growth can be introduced passed through a chilled, argon-flushed French pressure cell into the A. vinelandii genome by a marker-rescue procedure at 12,000 psi (82.7 MPa), followed by centrifugation at 17,000 where the mutation-vector DNA is used to transform a strain x g for 20 min at 40C. The extracts were then degassed under deleted for nifD to prototrophy (see Fig. 1). Mutations that argon and the MoFe and Fe protein activities were assayed result in a strictly Nif- phenotype can be introduced into the by acetylene reduction (20), with and without saturating A. vinelandii chromosome by the simultaneous transforma- amounts of the purified complementary protein. Specific tion of wild-type cells with mutation-vector DNA and puri- activities of these purified MoFe and Fe proteins were 2020 fied A. vinelandii chromosomal DNA that carries an and 1700 nmol of acetylene reduced per min per mg of uncharacterized rifampicin-resistance marker. Rifrtransform- protein, respectively. Protein concentrations were deter- ants are recovered by selection on rifampicin-containing mined by the biuret method (21). medium, and the coincident transfer (congression) ofthe Nif marker is identified by scoring transformants in the absence RESULTS AND DISCUSSION or presence of a fixed-nitrogen source (see ref. 16 for a detailed description of this procedure). Rationale for Amino Acid Replacements. Several indirect Cell Growth and Nitrogenase Derepression. Wild-type and approaches have been used in attempts to identify structur- mutant strains of A. vinelandii were cultured

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