Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase

We report the properties and reactivity of the catalytically active heterol ogous nitrogenase formed between the Fe protein from Clostridium pasteurian um (Cp2) and the MoFe protein from Klebsiella pneumoniae (Kp1). Under turno ver conditions, in the presence of MgATP, a stable 2:1 (Cp-2)(2)Kp1 electro n transfer complex is formed, in which the [4Fe-4S](+) centre of Cp2 is pro tected from chelation by alpha,alpha'-bipyridyl. However, the two Fe protei n-binding sites on Kp1 are not equivalent, since a 1:1 Cp2.Kp1 complex was isolated by gel filtration. The non-equivalence of the Fe protein binding s ites was also indicated by the inhibition pattern of Klebsiella nitrogenase by Cp2. The EPR spectrum of the isolated 1:1 Cp2.Kp1 complex showed an S=1 /2 signal characteristic of dithionite-reduced Cp2 and signals with g value s of 4.27, 3.73, 2.01 and 4.32, 3.63, 2.00 characteristic of the high- and low-pH forms of the FeMoco centre of Kp1, respectively. The unoccupied bind ing site of Kp1 of the isolated 1:1 Cp2.Kp1 complex was shown to be catalyt ically fully functional in combination with Kp2. In contrast to homologous nitrogenases, which require MgATP for detectable rates of electron transfer from the Fe protein, stopped-flow kinetic studies revealed that electron t ransfer from Cp2 to Kp1 occurred in the absence of MgATP with a rate consta nt of 0.065 s(-1). Subsequently, a slower transient decrease and restoratio n of absorption in the electronic spectrum in the 500-700 nm region was obs erved. These changes corresponded with those in the intensity of the S=3/2 EPR signal of the FeMoco centres of Kp1 and were consistent with the transi ent reduction of the FeMoco centre of Kp1 to an EPR-silent form, followed b y restoration of the signal at longer reaction times. These changes were no t associated with catalysis since no evolution of H-2 was detectable.