Characterization of a Variant Iron Protein of Nitrogenase That Is Impaired in Its Ability to Adopt the MgATP-induced Conformational Change

Abstract

An Azotobacter vinelandii nitrogenase iron protein mutant has been created which contains an alanine to glycine substitution at amino acid 157. The strain expressing this mutant Fe protein is able to grow under nitrogen-fixing conditions. This contrasts with an A. vinelandii strain described previously which is unable to grow under nitrogen-fixing conditions and which expresses an Fe protein variant that has an alanine to serine mutation at position 157. The A157S Fe protein was unable to support substrate reduction by nitrogenase because of an inability to undergo a required MgATP-induced conformational change. Although the A157G strain grows at 55% of the rate of the wild-type strain, purified A157G Fe protein is only able to support substrate reduction in in vitro assays at a rate that is approximately 20% of the rate supported by the wild-type Fe protein. Electron paramagnetic resonance, circular dichroism spectroscopies, and enzymatic activity data indicate that the A157G Fe protein adopts the correct conformation upon the binding of MgATP. However, kinetic studies using chelation show that this protein undergoes the conformational change more slowly than the wild-type protein. Thus, this mutant has lower activity because of an impaired ability to undergo this conformational change. Comparison of two available x-ray crystal structures of the native Fe protein alone and complexed with the MoFe protein has provided us with a model to explain the change in activity in alanine 157 mutants. Steric interactions with the side chain of residue 157 influence the protein's ability to undergo the initial MgATP-induced conformational change. In the case of the A157G mutant, however, once the correct conformation is attained, the protein can participate in all subsequent reactions including complex formation, electron transfer, and MgATP hydrolysis. Thus, the role of alanine 157 is to stabilize the proper initial conformation upon MgATP binding. An Azotobacter vinelandii nitrogenase iron protein mutant has been created which contains an alanine to glycine substitution at amino acid 157. The strain expressing this mutant Fe protein is able to grow under nitrogen-fixing conditions. This contrasts with an A. vinelandii strain described previously which is unable to grow under nitrogen-fixing conditions and which expresses an Fe protein variant that has an alanine to serine mutation at position 157. The A157S Fe protein was unable to support substrate reduction by nitrogenase because of an inability to undergo a required MgATP-induced conformational change. Although the A157G strain grows at 55% of the rate of the wild-type strain, purified A157G Fe protein is only able to support substrate reduction in in vitro assays at a rate that is approximately 20% of the rate supported by the wild-type Fe protein. Electron paramagnetic resonance, circular dichroism spectroscopies, and enzymatic activity data indicate that the A157G Fe protein adopts the correct conformation upon the binding of MgATP. However, kinetic studies using chelation show that this protein undergoes the conformational change more slowly than the wild-type protein. Thus, this mutant has lower activity because of an impaired ability to undergo this conformational change. Comparison of two available x-ray crystal structures of the native Fe protein alone and complexed with the MoFe protein has provided us with a model to explain the change in activity in alanine 157 mutants. Steric interactions with the side chain of residue 157 influence the protein's ability to undergo the initial MgATP-induced conformational change. In the case of the A157G mutant, however, once the correct conformation is attained, the protein can participate in all subsequent reactions including complex formation, electron transfer, and MgATP hydrolysis. Thus, the role of alanine 157 is to stabilize the proper initial conformation upon MgATP binding. The Fe protein of nitrogenase is one example of a large family of proteins (e.g. H-ras p21, recA, myosin) that have energy transduction mechanisms involving switching between conformational states upon nucleotide binding or hydrolysis (1Koonin E.V. J. Mol. Biol. 1993; 229: 1165-1174Crossref PubMed A. J. PubMed PubMed J. Biol. PubMed PubMed PubMed A. PubMed of the and structures of this family of proteins have two that in nucleotide which is the or the contains of the Fe protein and that in binding and of the PubMed PubMed J. PubMed PubMed PubMed The which in the Fe protein to a and to the in the family PubMed A. PubMed J. PubMed PubMed PubMed the nucleotide binding and in energy transduction this family of proteins the Fe protein has the of the only of the The two by the and by a to a that has two binding MgATP PubMed and PubMed Although the binding the of MgATP is approximately the PubMed the binding of MgATP to the Fe protein a large conformational change that of the of that PubMed this conformational change the Fe protein is able to to the MoFe protein of protein is an of two and two by the PubMed J. PubMed The MoFe has two of The that to the Fe protein PubMed The of in the MoFe protein is the and is a that to the and to PubMed J. PubMed PubMed J. PubMed J. 1993; PubMed J. 1993; PubMed Although the Fe protein alone can MgATP hydrolysis only the Fe protein to the MoFe protein. the energy by MgATP hydrolysis is by the Fe protein complex is However, the that the Fe protein is the only that can the MoFe protein in a that the can is to the energy transduction two the proteins that and undergo conformational the Fe protein is a The which is kinetic studies with an electron J. PubMed that the to the hydrolysis of two of MgATP to the of a electron the Fe protein to the MoFe protein PubMed with electron has that the of the Fe protein can by two to an all J. J. This a that the Fe protein is a because can two of electron in to two a of which is the binding of two of MgATP a large conformational change that a Although the of the Fe protein with MgATP has been is x-ray J. Biol. PubMed the of an Fe protein complex PubMed and J. Biol. PubMed J. Biol. PubMed PubMed 1993; PubMed PubMed that the conformational change interactions between the two of the Fe have a of the Fe protein which is at the and to the MgATP-induced conformational change J. Biol. PubMed This is in proteins or in protein is in the data of the Fe protein alone PubMed and in the Fe protein complex PubMed that this is of at the which residue to This the Fe protein is with the of the Fe protein in the studies have that mutation of to in a protein that can MgATP is unable to undergo the MgATP-induced conformational change. the mutation of this residue can the MgATP-induced conformational have to this and The mutation of to than to protein. the and of an A157G Fe protein that is all and or and of the Azotobacter vinelandii of the A. vinelandii the and of the was the was using the The was The was and was to the the The was at the the of Fe protein at this using the was using a This was two A. vinelandii the wild-type strain and the strain using a J. PubMed the of the is with the of variant Fe protein in native under the of native The of the the to mutation the of the strain in a strain that was able to grow under nitrogen-fixing conditions. subsequent was this and A157G A. vinelandii strain was in in a in under conditions. was to the to a of that the was The in was by the rate of the by and the of the of the the was an The using a The was with and was the initial of all subsequent with the of the of the circular dichroism in the of The variant Fe protein was purified described previously PubMed with and of was in of and was to a of and the by a two or at to a The was with and an at in a and a the the protein was by at The Fe protein was with a The was a and with The Fe was a and with a The was a and by in the using The purified Fe protein was by and was using the J. Biol. PubMed all in a under the in The a with an the was by one of two In one a of was to the protein a The protein was in the than and was the by a The of the protein by a described previously J. PubMed The to of to and of The protein was the of the and was to than was with The using a a activity was by under an and under an of and by the Fe and MoFe proteins at in the of a and in of and of the and of in of a was The of Fe protein to MoFe protein was and the protein was The was by the of Fe protein. the was by the of of The of was a using a or a The was that the was by the of of wild-type or A157G to a and The protein in the activity was using the J. Biol. PubMed and was to and the wild-type and A157G The was by a in the of and and the of the of the The assays of that the rate of was with to to of the by the of using a 1993; PubMed by An of the the activity was to protein. The nucleotide of a of this was by a and with using a at An of was to the of chelation J. Biol. PubMed PubMed was by a of and in a Fe protein was to a of and the The chelation was by the of MgATP to a of and The of the was by the of the at The ability of the Fe protein to with the wild-type MoFe protein was by the chelation in the of MoFe protein PubMed PubMed In this a was to the of The of and The Fe protein of nitrogenase is one example of a large family of proteins (e.g. H-ras p21, recA, myosin) that have energy transduction mechanisms involving switching between conformational states upon nucleotide binding or hydrolysis (1Koonin E.V. J. Mol. Biol. 1993; 229: 1165-1174Crossref PubMed A. J. PubMed PubMed J. Biol. PubMed PubMed PubMed A. PubMed of the and structures of this family of proteins have two that in nucleotide which is the or the contains of the Fe protein and that in binding and of the PubMed PubMed J. PubMed PubMed PubMed The which in the Fe protein to a and to the in the family PubMed A. PubMed J. PubMed PubMed PubMed Although the nucleotide binding and in energy transduction this family of proteins the Fe protein has the of the only of the The two by the and by a to a that has two binding MgATP PubMed and PubMed Although the binding the of MgATP is approximately the PubMed the binding of MgATP to the Fe protein a large conformational change that of the of that PubMed this conformational change the Fe protein is able to to the MoFe protein of protein is an of two and two by the PubMed J. PubMed The MoFe has two of The that to the Fe protein PubMed The of in the MoFe protein is the and is a that to the and to PubMed J. PubMed PubMed J. PubMed J. 1993; PubMed J. 1993; PubMed Although the Fe protein alone can MgATP hydrolysis only the Fe protein to the MoFe protein. the energy by MgATP hydrolysis is by the Fe protein complex is However, the that the Fe protein is the only that can the MoFe protein in a that the can is to the energy transduction two the proteins that and undergo conformational the Fe protein is a The which is kinetic studies with an electron J. PubMed that the to the hydrolysis of two of MgATP to the of a electron the Fe protein to the MoFe protein PubMed with electron has that the of the Fe protein can by two to an all J. J. This a that the Fe protein is a because can two of electron in to two a of which is the binding of two of MgATP a large conformational change that a Although the of the Fe protein with MgATP has been is x-ray J. Biol. PubMed the of an Fe protein complex PubMed and J. Biol. PubMed J. Biol. PubMed PubMed 1993; PubMed PubMed that the conformational change interactions between the two of the Fe protein. have a of the Fe protein which is at the and to the MgATP-induced conformational change J. Biol. PubMed This is in proteins or in protein is in the data of the Fe protein alone PubMed and in the Fe protein complex PubMed that this is of at the which residue to This the Fe protein is with the of the Fe protein in the studies have that mutation of to in a protein that can MgATP is unable to undergo the MgATP-induced conformational change. the mutation of this residue can the MgATP-induced conformational have to this and The mutation of to than to protein. the and of an A157G Fe protein that is all and or and of the Azotobacter vinelandii of the A. vinelandii the and of the was the was using the The was The was and was to the the The was at the the of Fe protein at this using the was using a This was two A. vinelandii the wild-type strain and the strain using a J. PubMed the of the is with the of variant Fe protein in native under the of native The of the the to mutation the of the strain in a strain that was able to grow under nitrogen-fixing conditions. subsequent was this and A157G A. vinelandii strain was in in a in under conditions. was to the to a of that the was The in was by the rate of the by and the of the of the the was an The using a The was with and was the initial of all subsequent with the of the of the circular dichroism in the of The variant Fe protein was purified described previously PubMed with and of was in of and was to a of and the by a two or at to a The was with and an at in a and a the the protein was by at The Fe protein was with a The was a and with The Fe was a and with a The was a and by in the using The purified Fe protein was by and was using the J. Biol. PubMed all in a under the in The a with an the was by one of two In one a of was to the protein a The protein was in the than and was the by a The of the protein by a described previously J. PubMed The to of to and of The protein was the of the and was to than was with The using a a activity was by under an and under an of and by the Fe and MoFe proteins at in the of a and in of and of the and of in of a was The of Fe protein to MoFe protein was and the protein was The was by the of Fe protein. the was by the of of The of was a using a or a The was that the was by the of of wild-type or A157G to a and The protein in the activity was using the J. Biol. PubMed and was to and the wild-type and A157G The was by a in the of and and the of the of the The assays of that the rate of was with to to of the by the of using a 1993; PubMed by An of the the activity was to protein. The nucleotide of a of this was by a and with using a at An of was to the of chelation J. Biol. PubMed PubMed was by a of and in a Fe protein was to a of and the The chelation was by the of MgATP to a of and The of the was by the of the at The ability of the Fe protein to with the wild-type MoFe protein was by the chelation in the of MoFe protein PubMed PubMed In this a was to the of The of and all and or and of the Azotobacter vinelandii of the A. vinelandii the and of the was the was using the The was The was and was to the the The was at the the of Fe protein at this using the was using a This was two A. vinelandii the wild-type strain and the strain using a J. PubMed the of the is with the of variant Fe protein in native under the of native The of the the to mutation the of the strain in a strain that was able to grow under nitrogen-fixing conditions. subsequent was this and A157G A. vinelandii strain was in in a in under conditions. was to the to a of that the was The in was by the rate of the by and the of the of the the was an The using a The was with and was the initial of all subsequent with the of the of the circular dichroism in the of The variant Fe protein was purified described previously PubMed with and of was in of and was to a of and the by a two or at to a The was with and an at in a and a the the protein was by at The Fe protein was with a The was a and with The Fe was a and with a The was a and by in the using The purified Fe protein was by and was using the J. Biol. PubMed all in a under the in The a with an the was by one of two In one a of was to the protein a The protein was in the than and was the by a The of the protein by a described previously J. PubMed The to of to and of The protein was the of the and was to than was with The using a a activity was by under an and under an of and by the Fe and MoFe proteins at in the of a and in of and of the and of in of a was The of Fe protein to MoFe protein was and the protein was The was by the of Fe protein. the was by the of of The of was a using a or a The was that the was by the of of wild-type or A157G to a and The protein in the activity was using the J. Biol. PubMed and was to and the wild-type and A157G The was by a in the of and and the of the of the The assays of that the rate of was with to to of the by the of using a 1993; PubMed by An of the the activity was to protein. The nucleotide of a of this was by a and with using a at An of was to the of chelation J. Biol. PubMed PubMed was by a of and in a Fe protein was to a of and the The chelation was by the of MgATP to a of and The of the was by the of the at The ability of the Fe protein to with the wild-type MoFe protein was by the chelation in the of MoFe protein PubMed PubMed In this a was to the of The of and and of the Azotobacter vinelandii of the A. vinelandii the and of the was the was using the The was The was and was to the the The was at the the of Fe protein at this using the was using a This was two A. vinelandii the wild-type strain and the strain using a J. PubMed the of the is with the of variant Fe protein in native under the of native The of the the to mutation the of the strain in a strain that was able to grow under nitrogen-fixing conditions. subsequent was this and A157G A. vinelandii strain was in in a in under conditions. was to the to a of that the was The in was by the rate of the by and the of the of the the was an The using a The was with and was the initial of all subsequent with the of the of the circular dichroism in the of The variant Fe protein was purified described previously PubMed with and of was in of and was to a of and the by a two or at to a The was with and an at in a and a the the protein was by at The Fe protein was with a The was a and with The Fe was a and with a The was a and by in the using The purified Fe protein was by and was using the J. Biol. PubMed all in a under the in The a with an the was by one of two In one a of was to the protein a The protein was in the than and was the by a The of the protein by a described previously J. PubMed The to of to and of The protein was the of the and was to than was with The using a a activity was by under an and under an of and by the Fe and MoFe proteins at in the of a and in of and of the and of in of a was The of Fe protein to MoFe protein was and the protein was The was by the of Fe protein. the was by the of of The of was a using a or a The was that the was by the of of wild-type or A157G to a and The protein in the activity was using the J. Biol. PubMed and was to and the wild-type and A157G The was by a in the of and and the of the of the The assays of that the rate of was with to to of the by the of using a 1993; PubMed by An of the the activity was to protein. The nucleotide of a of this was by a and with using a at An of was to the of chelation J. Biol. PubMed PubMed was by a of and in a Fe protein was to a of and the The chelation was by the of MgATP to a of and The of the was by the of the at The ability of the Fe protein to with the wild-type MoFe protein was by the chelation in the of MoFe protein PubMed PubMed In this a was to the of The of and and of the Azotobacter vinelandii of the A. vinelandii the and of the was the was using the The was The was and was to the the The was at the the of Fe protein at this using the was using a This was two A. vinelandii the wild-type strain and the strain using a J. PubMed the of the is with the of variant Fe protein in native under the of native The of the the to mutation the of the strain in a strain that was able to grow under nitrogen-fixing conditions. subsequent was this of the A. vinelandii the and of the was the was using the The was The was and was to the the The was at the the of Fe protein at this using the was using a This was two A. vinelandii the wild-type strain and the strain using a J. PubMed the of the is with the of variant Fe protein in native under the of native The of the the to mutation the of the strain in a strain that was able to grow under nitrogen-fixing conditions. subsequent was this and A157G A. vinelandii strain was in in a in under conditions. was to the to a of that the was The in was by the rate of the by and the of the of the the was an The using a The was with and was the initial of all subsequent with the of the of the circular dichroism in the of The variant Fe protein was purified described previously PubMed with and of was in of and was to a of and the by a two or at to a The was with and an at in a and a the the protein was by at The Fe protein was with a The was a and with The Fe was a and with a The was a and by in the using The purified Fe protein was by and was using the J. Biol. PubMed The A157G A. vinelandii strain was in in a in under conditions. was to the to a of that the was The in was by the rate of the by and the of the of the the was an The using a The was with and was the initial of all subsequent with the of the of the circular dichroism in the of The variant Fe protein was purified described previously PubMed with and of was in of and was to a of and the by a two or at to a The was with and an at in a and a the the protein was by at The Fe protein was with a The was a and with The Fe was a and with a The was a and by in the using The purified Fe protein was by and was using the J. Biol. PubMed all in a under the in The a with an the was by one of two In one a of was to the protein a The protein was in the than and was the by a The of the protein by a described previously J. PubMed The to of to and of The protein was the of the and was to than was with The using a a all in a under the in The a with an the was by one of two In one a of was to the protein a The protein was in the than and was the by a The of the protein by a described previously J. PubMed The to of to and of The protein was the of the and was to than was with The using a a activity was by under an and under an of and by the Fe and MoFe proteins at in the of a and in of and of the and of in of a was The of Fe protein to MoFe protein was and the protein was The was by the of Fe protein. the was by the of of The of was a using a or a The was that the was by the of of wild-type or A157G to a and The protein in the activity was using the J. Biol. PubMed and was to and the wild-type and A157G The was by a in the of and and the of the of the The assays of that the rate of was with to to of the by the of using a 1993; PubMed by An of the the activity was to protein. The nucleotide of a of this was by a and with using a at An of was to the of activity was by under an and under an of and by the Fe and MoFe proteins at in the of a and in of and of the and of in of a was The of Fe protein to MoFe protein was and the protein was The was by the of Fe protein. the was by the of of The of was a using a or a The was that the was by the of of wild-type or A157G to a and The protein in the activity was using the J. Biol. PubMed and was to and the wild-type and A157G The was by a in the of and and the of the of the The assays of that the rate of was with to to of the by the of using a 1993; PubMed by An of the the activity was to protein. The nucleotide of a of this was by a and with using a at An of was to the of chelation J. Biol. PubMed PubMed was by a of and in a Fe protein was to a of and the The chelation was by the of MgATP to a of and The of the was by the of the at The ability of the Fe protein to with the wild-type MoFe protein was by the chelation in the of MoFe protein PubMed PubMed In this a was to the of The of and The chelation J. Biol. PubMed PubMed was by a of and in a Fe protein was to a of and the The chelation was by the of MgATP to a of and The of the was by the of the at The ability of the Fe protein to with the wild-type MoFe protein was by the chelation in the of MoFe protein PubMed PubMed In this a was to the of The of and the A157S Fe the of the Fe protein complex crystal and and the of this