Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation

Emilio Jimenez-Vicente,Zhi-Yong Yang,W Keith Ray,Carlos Echavarri-Erasun,Valerie L Cash,Luis M Rubio,Lance C Seefeldt,Dennis R Dean

doi:10.1074/jbc.ra118.002994

Abstract

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.

Highlights

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia
Given the critical importance of the associated metal-containing clusters to nitrogenase catalysis, an understanding of all factors contributing to the robust assembly of MoFe protein containing intact FeMo-cofactor and P-cluster is of considerable interest
Nitrogen-fixing bacterium Azotobacter vinelandii, there are 55 genes whose expression is increased in response to a demand for fixed nitrogen when cultured under molybdenum-sufficient conditions (Fig. 2) [11, 16]

Summary

Introduction

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. The MoFe protein is an ␣2␤2 heterotetramer harboring two catalytic units and two pairs of complex metal-containing clusters, both of which, so far, are unique in biological systems [4, 5] (Fig. 1) One of these is a [7Fe-9S-Mo-C-homocitrate] species called FeMo-cofactor, and the other is an [8Fe-7S] cluster designated the P-cluster. Given the critical importance of the associated metal-containing clusters to nitrogenase catalysis, an understanding of all factors contributing to the robust assembly of MoFe protein containing intact FeMo-cofactor and P-cluster is of considerable interest. The construction of strains described here permitted evaluation of the suggestion that an apo-MoFe protein containing P-cluster precursors, but not FeMo-cofactor, has the capacity for Fe protein-dependent substrate reduction [26]

Methods

Results

Conclusion