Substrate pathways in the nitrogenase MoFe protein by experimental identification of small molecule binding sites

Abstract

In the nitrogenase molybdenum-iron (MoFe) protein, we have identified five potential substrate access pathways from the protein surface to the FeMo-cofactor (the active site) or the P-cluster using experimental structures of Xe pressurized into MoFe protein crystals from Azotobacter vinelandii and Clostridium pasteurianum. Additionally, all published structures of the MoFe protein, including those from Klebsiella pneumoniae, were analyzed for the presence of nonwater, small molecules bound to the protein interior. Each pathway is based on identification of plausible routes from buried small molecule binding sites to both the protein surface and a metallocluster. Of these five pathways, two have been previously suggested as substrate access pathways. While the small molecule binding sites are not conserved among the three species of MoFe protein, residues lining the pathways are generally conserved, indicating that the proposed pathways may be accessible in all three species. These observations imply that there is unlikely a unique pathway utilized for substrate access from the protein surface to the active site; however, there may be preferred pathways such as those described here.

Scheme 1. Standard Model for Biological Nitrogen Fixation

Figure 1

Ribbon representation of Cp1 with the locations indicated for all Xe, PRL, and other small molecule binding sites. The α- and β-subunits are colored in green and cyan, respectively. The FeMo-cofactor, P-cluster, Fe16, and small molecules are displayed as small spheres colored by element. Av1 and Cp1 Xe sites are shown in large blue and magenta spheres, respectively. Binding sites observed in Av1 and Kp1 structures are superposed onto the Cp1 structure. (Cp1-Xe PDB ID: 4WN9; Av1-Xe PDB ID: 4WNA).

Figure 2

Ribbon representation of Av1 illustrating the channels and pathways discussed in this study, as viewed from two perpendicular orientations, one of which is scaled down. The α-subunits are colored green and magenta, and the β-subunits are colored cyan and yellow. The cofactors are shown in ball-and-stick representation colored by element. Pathways were calculated using CAVER and are displayed as surfaces within the protein structure. This figure was created in PyMOL.

Figure 3

(a) The AI/IS pathway. From the protein surface to the Xe binding site, the AI and IS pathways follow the light purple and cyan pathways, respectively. From the Xe binding site to the FeMo-cofactor, the pathways (slate blue) are the same. Substrates may penetrate the protein surface at the Av1-Xe2 binding site following the light purple pathway or the cyan pathway. Upon reaching the Av1-Xe1 binding site, substrates may continue toward the FeMo-cofactor following the slate blue pathway. The Fe atoms accessed on the FeMo-cofactor by this pathway are labeled. Av1-Xe1 and Av1-Xe2 are displayed as large blue spheres. (b) The AII pathway (magenta and light pink surfaces). Substrates may reach the Cp1-Xe3 binding site following the light pink pathway and then continue toward the FeMo-cofactor following the magenta pathway. Cp1-Xe3 is displayed as a large magenta sphere. In both figures, residues lining the pathways are labeled. The α, β, and β′ subunits are shown in green, cyan, and yellow, respectively.

Figure 4

(a) PRL binds in an arm of the center channel (brown surface) that reaches toward the cofactors. The arm terminates before reaching the FeMo-cofactor, so substrates must continue to the cofactors within the protein scaffold. The most likely pathway (in terms of size) is between the α- and β-subunits. Branching from this pathway, substrates may either head toward the FeMo-cofactor (forest green surface, pathway BI) or toward the P-cluster (green surface, pathway CI). (b) A close-up view of the BI and CI pathways leading to the FeMo-cofactor and P-cluster, respectively. In both figures, the α, β, and β′ subunits are shown in green, cyan, and yellow, respectively. Residues lining the pathway are shown in sticks and labeled. The cofactors and PRL are displayed as spheres colored by element.

Figure 5

DI pathway (orange surface). The EDO and IMD molecules bind in the DI pathway/interstitial channel that connects the protein surface to the Fe2,3,6,7 and Fe3,4,5,7 FeMo-cofactor faces (orange surface). The CO and S bind in a channel that extends from the DI pathway (purple surface). The α, β, and β′ subunits are shown in green, cyan, and yellow, respectively. Residues lining the pathway are shown in sticks and labeled. The substrates and FeMo-cofactor are displayed as spheres colored by element. The IMD and EDO molecules are superposed in the figure because the binding sites directly overlap.