Formation of a Tight 1:1 Complex of Clostridium pasteurianum Fe Protein-Azotobacter Vinelandii MoFe Protein: Evidence for Long-Range Interactions between the Fe Protein Binding Sites during Catalytic Hydrogen Evolution † Thomas A. Clarke, Silvana Maritano, and Robert R. Eady* Nitrogen Fixation Laboratory, John Innes Centre, Colney, Norwich NR4 7UH, U.K. ReceiVed February 8, 2000; ReVised Manuscript ReceiVed June 6, 2000 ABSTRACT: It has been well documented that the combination of the MoFe protein of Azotobacter Vinelandii nitrogenase (Av1) with the Fe protein (Cp2) from Clostridium pasteurianum nitrogenase produces an inactive, stable complex. However, we report that this heterologous nitrogenase has a low level of activity for H 2 evolution, with a specific activity of 12 nmol min -1 mg -1 of Av1. This activity does not arise from contaminating hydrogenase since it required the presence of both Cp2 and Av1 and showed saturation kinetics when increasing amounts of Cp2 were added to the assay. Incubation of the two proteins at a 4:1 Cp2:Av1 ratio in the absence of MgATP followed by analytical gel filtration showed, surprisingly, that the stoichiometry of the isolated complex was Av1‚Cp2 instead of Av1‚(Cp2) 2 as determined previously. The presence of MgATP in the elution buffer did not change the elution profile of the complex. The hydrodynamic radius of the isolated complex determined by dynamic light scattering was 5.93 ( 0.14 nm, intermediate between Av1 and a stable 2:1 nitrogenase complex, consistent with a 1:1 assignment for the Av1‚Cp2 complex. When assayed with Av2, the isolated Av1‚Cp2 complex showed full half-site reactivity with a specific activity of 750 nmol of C 2 H 2 reduced min -1 mg -1 of Av1. The EPR spectrum of the isolated complex showed the Cp2 to be oxidized and the Av1 to retain the S ) 3 / 2 signal characteristic of FeMoco. In the presence of MgATP, under turnover conditions at a 2:1 ratio of Cp2:Av1, the [4Fe-4S] center of Cp2 was protected from the chelator 2,2′-bipyridyl. This is consistent with the formation of a tight 2:1 complex of Av1‚(Cp2) 2 which is more stable than the homologous Cp nitrogenase. Assuming that the Lowe-Thorneley model for nitrogenase applies and that a rate-limiting dissociation of the complex is required for H 2 evolution, then with a rate of 0.032 s -1 the 1:1 complex is too stable to be involved in catalysis. The differences in the stability of the 2:1 and 1:1 complexes indicate cooperativity between the Fe protein binding sites of Av1, which structural data show to be separated by 105 Å. On the basis of these observations, we propose a model for nitrogenase catalysis in which the stable 1:1 complex formed between oxidized Fe protein and the one-electron-reduced MoFe protein plays an essential role. In this scheme, the two Fe protein binding sites of the MoFe protein alternately bind and release Fe protein in a shuttle mechanism associated with long-range conformational changes in the MoFe protein. Nitrogenase, the enzyme system responsible for biological nitrogen fixation, catalyzes the reduction of dinitrogen to ammonia via the MgATP-dependent reaction: Three related nitrogenase systems have been characterized, the first containing Fe and Mo, which is widespread and the most extensively studied (1, 2), a second utilizing Fe and V, and a third based on Fe (see ref 3). An unrelated superoxide-dependent Mo-containing system isolated from Streptomyces thermoautotrophicus has recently been de- scribed (4). The relative contribution that these systems make to global nitrogen cycling has yet to be assessed; however, it is likely that the conventional Mo-containing enzyme, which appears to be present in all nitrogen-fixing organisms other than S. thermoautotrophicus, is a major route for nitrogen fixation and is the enzyme studied in the present work. This Mo-containing enzyme is comprised of two separate component proteins: an Fe protein, responsible for MgATP-driven electron transfer, and a larger MoFe protein, which contains the catalytic site for nitrogen reduction. The electrons for the reaction are donated to the Fe protein from flavodoxin or ferredoxin in vivo and usually sodium dithion- ite in vitro (1-3). In addition to N 2 , nitrogenase will reduce a number of alternative substrates including C 2 H 2 , and in the absence of a reducible substrate protons are reduced to H 2 . Mo nitrogenases are a highly conserved enzyme family † Financial support for this work was provided by the Biotechnology and Biological Sciences Research Council as part of the Competitive Strategic Grant to the John Innes Centre and a research studentship to T.A.C. and by the European Union for a Marie Curie fellowship (ERBFMBICT982909) to S.M. * Address correspondence to this author: e-mail, robert.eady@ bbsrc.ac.uk; tel, 44 1603 450728; fax, 44 1603 450018. N 2 + 8H + + 8e - + 16MgATP f 2NH 3 + H 2 + 16MgADP + 16P i 11434 Biochemistry 2000, 39, 11434-11440 10.1021/bi0002939 CCC: $19.00 © 2000 American Chemical Society Published on Web 08/18/2000