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

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.

Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology

“…It is possible that this represents a kinetic effect since nifW and nifZ mutants delay the induction of nitrogenase activity in K. oxytoca ( 29 ). Recently, it has been demonstrated that NifW and NifZ bind to immature forms of apo-NifDK and are likely to function as assembly factors required for P-cluster maturation ( 47 ). Since diazotrophic growth is slower in nifW mutants, it has been suggested that either the function of this gene can be partially complemented by another gene or nifW plays a kinetic role in MoFe protein maturation.…”

The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters

“…A variety of assembly factors are either required for or assist in the formation of the metalloclusters necessary for nitrogenase activity (10, 11, 13). For example, NifH (Fe protein), NifU, NifS, NifB, NifEN, and NifV represent the minimum set of proteins required for in vivo formation of FeMo-cofactor, which is separately synthesized and then inserted into an apo-form of the MoFe protein already containing intact P-clusters (Fig.…”

“…1) (14, 15). Although details of P-cluster formation are not yet known, three accessory proteins, NafH, NifW, and NifZ, as well as NifH have been shown to sequentially and differentially interact with MoFe protein during the process of P-cluster formation/maturation (13). Inactivation of NifZ, which functions after NifW, produces MoFe protein containing some FeMo-cofactor, as well as a mixture of both mature and immature P-clusters.…”

Natural and Engineered Electron Transfer of Nitrogenase

“…TOFs for this system reached maxima of 0.02 (for CH 4 production from CN − , single time point) and 0.17 (for NH 3 production from CN − , single time point). Interestingly, the authors observed a~2.5-fold increase in activity when using the ∆nifH MoFe protein vs. the ∆nifB MoFe protein; Jimenez-Vicente et al recently demonstrated that the Fe protein of the V-nitrogenase system (encoded by vnfH and typically repressed in the presence of Mo) can substitute the NifH Fe protein in ∆nifH strains, which could explain the observed elevated activities [161]. In addition to the Mo-nitrogenase system, Eu(II) complexes have also been utilized to deliver electrons to the alternative V-nitrogenase system for MgATP-independent substrate reduction [162].…”