Abstract
Unlike wild type, certain Mo-dependent nitrogenases, which are expressed in non-N2-fixing mutant strains of Azotobacter vinelandii and have single amino acid substitutions within a region of the MoFe protein alpha-subunit proposed to encompass an FeMo cofactor-binding domain, are able to catalyze the reduction of acetylene by both two and four electrons to yield ethylene and ethane, respectively (Scott, D. J., May, H. D., Newton, W. E., Brigle, K. E., and Dean, D. R. (1990) Nature 343, 188-190). Although the V-dependent nitrogenase is also able to catalyze the reduction of acetylene to the same two- and four-electron products (Dilworth, M. J., Eady, R. R., Robson, R. L., and Miller, R. W. (1987) Nature 327, 167-168), we find that ethane formation from acetylene catalyzed by the altered Mo-dependent nitrogenases occurs by a different mechanism, which is distinguished by: (i) an increased sensitivity to CO; (ii) the absence of a lag; and (iii) no temperature dependence of product distribution among ethylene and ethane during acetylene reduction. An altered MoFe protein, which was purified from one such mutant strain having the alpha-subunit glutaminyl 191 residue substituted by lysyl, exhibited both a changed S = 3/2 EPR spectrum and changes in the distribution of electrons to various products when compared to wild type. Also, unlike wild type, this altered MoFe protein catalyzed proton reduction that is inhibited by carbon monoxide (CO). Because proton reduction catalyzed by a nitrogenase that has a FeMo cofactor with citrate rather than homocitrate as its organic constituent (Liang, J., Madden, M., Shah, V. K., and Burris, R. H. (1990) Biochemistry 29, 8577-8581) is also inhibited by CO, the possibility arose that changes in the polypeptide environment of FeMo cofactor might have caused a rearrangement in its molecular structure or composition. However, this possibility was ruled out by biochemical reconstitution studies (using FeMo cofactor isolated from both the wild-type and altered MoFe proteins), which were monitored by EPR spectroscopy and resulting catalytic activity.
Highlights
LeComte, 1966), which consists of the Fe protein and theMoFe protein.The Fe protein factor-binding domain,are able to catalyze the reduc-is an ATP-binding, one-electron donor to the MoFe protein dpRMdatyinieruo.eoapcdnlyd(tedi,Mu1nooHecf9dnitt9e.lhsoalnD0eyfc(t)r.leaD,enNtcNnyRiiealtelwe.tretyowaunWolgnretere.odetnbnhn(eeya,3,t1Msb4Wh9teo3a.o8.t,niJh7sEe.t)t.,ha,NwrEle1Besoaa8osrtda8spiugaany-erblmd,1ceel9tRf,eeio03v.K)t2ueRot..7Arlw.Ey,e,clo.lta(,Reh-Stacooactnulba1roydgsno6tzohtdn7De,ns,ettt1fhaDhRoo6nee..u8,rwLr)JVDe,..ee--., l e cJecAus1totqap9rmrhtuoo7aanoi3ntnlvens)ya.swogzIl(nenhetfsihnocetetrththshseaeaaetphnlar.rred,ereeesdsac1euuen9nNcnb7catts2Peinto;rar,onepaSefvtrrmMeioooe-ftbiwgbotiahAi,ncnssTevde,enaPtiaenrv,naigiaDedlr.tsos,eyuinaat1icenmot9e(afa7etsbyn3)snllm;detea,MnsrJaMeoealolu,cloworrom-ctcbhneeoasinitocltoseerhfnodcorun,ge(ladeeO1ernue9trcsam9ruisan2eebleg-).,-. find that ethane formation from acetylene catalyzed spectively reducedto yield ammonia, Hz, anedthylene
An altered MoFe protein, which was puri- of MgATP arehydrolyzed and a single electron is transferred fied from one such mutant strain having the a-subufnroitm theFeproteintothe MoFe protein(Hagemanand glutaminyl 191 residue substituted bylysyl, exhibited Burris, 1980; Lowe and Thorneley, 1984)
In aprevious study (Scott et al, 1990), we showed that the polypeptide
Summary
Crude Extracts-Maximal Fe protein and MoFe protein activities of crude extracts were measured by the acetylene-reduction assay by titration with increasing amounts of either purified wild-type MoFe protein or purified wild-type Fe protein, respectively. The effect of temperature on electron flux through nitrogenase in crude extracts of DJ178 (D195H-N) and wild-type cells was MATERIALS ANDMETHODS assessed by performing the usual acetylene-reduction assays in the presence of saturating levels of purified, wild-type Fe protein, hut at Mutant StrainConstruction a temperatureof 10,20,30, or 40 "C.The linearity of the rates a t the various temperatures was established by measuring activities over a Methods for site-directed mutagenesis, gene replacement, and the isolation of mutantstrains were performed as described or cited previously (Brigle et al, 1987a, 1987b).The isolation of strains DJ42, DJ64, DJ178, DJ255, and DJ357 was previously reported (Brigle et al, 1987a, 1987b; Scott et al, 1990). Protein concentrations were determined by the biuret method (Gornall etal., 1948)
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