Abstract
Although the nitrogen-fixing enzyme nitrogenase critically requires both a reductase component (Fe protein) and a catalytic component, considerably more work has focused on the latter species. Properties of the catalytic component, which contains two highly complex metallocofactors and catalyzes the reduction of N2 into ammonia, understandably making it the “star” of nitrogenase. However, as its obligate redox partner, the Fe protein is a workhorse with multiple supporting roles in both cofactor maturation and catalysis. In particular, the nitrogenase Fe protein utilizes nucleotide binding and hydrolysis in concert with electron transfer to accomplish several tasks of critical importance. Aside from the ATP-coupled transfer of electrons to the catalytic component during substrate reduction, the Fe protein also functions in a maturase and insertase capacity to facilitate the biosynthesis of the two-catalytic component metallocofactors: fusion of the [Fe8S7] P-cluster and insertion of Mo and homocitrate to form the matured [(homocitrate)MoFe7S9C] M-cluster. These and key structural-functional relationships of the indispensable Fe protein and its complex with the catalytic component will be covered in this review.
Highlights
The enzyme molybdenum (Mo)-nitrogenase is capable of facilitating the biological fixation of atmospheric dinitrogen into bioavailable nitrogen sources at ambient temperature and pressure [1] in the following reaction: N2 + 8e− + 16MgATP + 8H+ → 2NH3 + H2 + 16MgADP + 16Pi (1)where ATP is adenosine triphosphate, ADP is adenosine diphosphate, and Pi is inorganic phosphate.The catalytic enzyme component that is responsible for the N2 reduction activity in Azotobacter vinelandii is the gene product NifDK, which is commonly known as the MoFe protein.NifDK is an α2 β2 tetramer that houses two sets of complex metallocofactors: an [Fe8 S7 ] cofactor known as the P-cluster and a [MoFe7 S9 C-homocitrate] cofactor called the M-cluster (Figure 1)
Biochemical maturation studies with NifEN have shown that in order to mature the bound L-cluster to the M-cluster, the following components, at minimum, must be incubated with this protein: dithionite, NifH, MgATP, molybdate (MoO4 2− ), and R-homocitrate [12]. This incubation mixture unambiguously leads to accumulation of the M-cluster on NifEN, as established by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies [13]
When ATP is bound to NifH, Fe is rapidly removed from the metallocluster in the presence of these same chelating agents [54,55,56]. These results indicate that limited chelator access to the [Fe4 S4 ] cluster takes place in the absence of MgATP, but with nucleotide-bound NifH, the cluster is rendered more solvent exposed
Summary
The enzyme molybdenum (Mo)-nitrogenase is capable of facilitating the biological fixation of atmospheric dinitrogen into bioavailable nitrogen sources at ambient temperature and pressure [1] in the following reaction: N2 + 8e− + 16MgATP + 8H+ → 2NH3 + H2 + 16MgADP + 16Pi (1). In addition to NifDK, catalysis by the enzyme requires a reductase component called NifH (or Fe protein), which is a homodimeric protein that contains a [Fe4 S4 ] cluster and nucleotide binding sites [2]. There are three primary recognized functions: (1) Mo and homocitrate insertase for the maturation of an 8Fe precursor to the M-cluster; (2) reductase to facilitate P-cluster formation for the maturation of an 8Fe precursor to the M-cluster; (2) reductase to facilitate P-cluster formation on NifDK; and (3) essential electron transfer partner to NifDK for nitrogen fixation catalysis. The attribution of these functions to the Fe protein is best illustrated by strains of A. vinelandii that had the gene encoding for nifH deleted [5] These ΔnifH mutant strains express a NifDK that (1).
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