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 c omponent of nitrogenase. Both MgATP molecules are hydrolyzed to MgADP and P -i in reactions coupled to the transfer of one electron into the MoFe prote in component, As an approach to assess the contributions of individual ATP binding sites, a heterodimeric Fe protein was produced that has an Asn subs tituted for residue 39 in the ATP binding domain in one subunit, while the normal Asp(39) residue within the other subunit remains unchanged. Separati on of the heterodimeric Fe protein from a mixed population with homodimeric Fe proteins contained in crude extracts was accomplished by construction o f a seven His tag on one subunit and a differential immobilized-metal-affin ity chromatography technique. Three forms of the Fe protein (wild-type homo dimeric Fe protein [Asp(39)/Asp(39)], altered homodimeric Fe protein [Asn(3 9)/Asn(39)], and heterodimeric Fe protein [Asp(39)/Asn(39)]) were compared on the basis of the biochemical and biophysical changes elicited by nucleot ide binding. Among those features examined were the MgATP- and MgADP-induce d protein conformational changes that are manifested by the susceptibility of the [4Fe-4S] cluster to chelation and by alterations in the electron par amagnetic resonance, circular dichroism, and midpoint potential of the [4Fe -4S] cluster. The results indicate that changes in the [4Fe-4S] cluster cau sed by nucleotide binding are the result of additive conformational changes contributed by the individual subunits. The [Asp(39)/Asn(39)] Fe protein d id not support substrate reduction activity but did hydrolyze MgATP and sho wed MgATP-dependent primary electron transfer to the MoFe protein. These re sults support a model where each MgATP site contributes to the rate acceler ation of primary electron transfer, but both MgATP sites must be functionin g properly for substrate reduction. Like the altered homodimeric [Asn(39)/A sn(39)] Fe protein, the heterodimeric [Asp(39)/Asn(39)] Fe protein was foun d to form a high affinity complex with the MoFe protein, revealing that alt eration on one subunit is sufficient to create a tight complex.