Construction and characterization of a heterodimeric iron protein: defining roles for adenosine triphosphate in nitrogenase catalysis

One molecule of MgATP binds to each subunit of the homodimeric Fe protein component of nitrogenase. Both MgATP molecules are hydrolyzed to MgADP and P(i) in reactions coupled to the transfer of one electron into the MoFe protein component. As an approach to assess the contributions of individual ATP binding sites, a heterodimeric Fe protein was produced that has an Asn substituted for residue 39 in the ATP binding domain in one subunit, while the normal Asp(39) residue within the other subunit remains unchanged. Separation of the heterodimeric Fe protein from a mixed population with homodimeric Fe proteins contained in crude extracts was accomplished by construction of a seven His tag on one subunit and a differential immobilized-metal-affinity chromatography technique. Three forms of the Fe protein (wild-type homodimeric Fe protein Asp(39)/Asp(39)], altered homodimeric Fe protein Asn(39)/Asn(39)], and heterodimeric Fe protein Asp(39)/Asn(39)]) were compared on the basis of the biochemical and biophysical changes elicited by nucleotide binding. Among those features examined were the MgATP- and MgADP-induced protein conformational changes that are manifested by the susceptibility of the 4Fe-4S] cluster to chelation and by alterations in the electron paramagnetic resonance, circular dichroism, and midpoint potential of the 4Fe-4S] cluster. The results indicate that changes in the 4Fe-4S] cluster caused by nucleotide binding are the result of additive conformational changes contributed by the individual subunits. The Asp(39)/Asn(39)] Fe protein did not support substrate reduction activity but did hydrolyze MgATP and showed MgATP-dependent primary electron transfer to the MoFe protein. These results support a model where each MgATP site contributes to the rate acceleration of primary electron transfer, but both MgATP sites must be functioning properly for substrate reduction. Like the altered homodimeric Asn(39)/Asn(39)] Fe protein, the heterodimeric Asp(39)/Asn(39)] Fe protein was found to form a high affinity complex with the MoFe protein, revealing that alteration on one subunit is sufficient to create a tight complex.