Computational Insights into Five- versus Six-Coordinate Iron Center in Ferrous Soybean Lipoxygenase

Abstract

Soybean lipoxygenase (SLO) serves as a prototype for fundamental understanding of hydrogen tunneling in enzymes. Its reactivity depends on the active site structure around a mononuclear, nonheme iron center. The available crystal structures indicate five-coordinate iron, while magnetic circular dichroism experiments suggest significant populations of both five-coordinate (5C) and six-coordinate (6C) iron in ferrous SLO. Quantum mechanical calculations of gas phase models produce only 6C geometries. Herein mixed quantum mechanical/molecular mechanical (QM/MM) calculations are employed to identify and characterize the 5C and 6C geometries. These calculations highlight the importance of the protein environment, particularly two Gln residues in a hydrogen-bonding network with Asn694, the ligand that can dissociate. This hydrogen-bonding network is similar in both geometries, but twisting of a dihedral angle in Asn694 moves its oxygen away from the iron in the 5C geometry. These insights are important for future simulations of SLO.

Figure 1

Depiction of the two gas phase model systems studied in this paper. The Mod-S model includes the Fe center and His499, His504, His690, Ile839, Asn694, and a bound water molecule. The Mod-L model also includes Gln495 and Gln697 and replaces the His residues by NH₃ and the Ile residue by formate.

Figure 2

SLO apo enzyme with QM region depicted in purple (left), and a magnified view of the QM region with the iron in pink and the two Gln residues in orange (right).

Figure 3

(A) Active site obtained from the crystal structure 3PZW, including definitions of the potential hydrogen bonds involving Gln495, Gln697, and Asn694 and the C–N–O angle α. (B) Superimposition of the 5C and 6C structures of ferrous SLO determined by QM/MM geometry optimization using the QM-L quantum region and the B3LYP functional. The Fe–O694 distance is 2.85 Å in the 5C structure (dark colors, black dashed line) and is 2.36 Å in the 6C structure (light colors, gray dashed line). The two hydrogen bonds HB1 and HB2 are depicted by dashed lines, and the associated distances and angles in the optimized geometries are given in Table S5.

Figure 4

Orientation of the ligated water molecule depicted by superimposition of the 5C and 6C structures of ferrous SLO determined by QM/MM geometry optimization using the QM-L quantum region and the B3LYP functional. The distance between OD1(Asn694) and the ligated water O was 2.68 Å and 2.72 Å for the 5C and 6C structures, respectively. The angle formed by OD1(Asn694), the ligated water H, and the ligated water O was 135° and 123° for the 5C and 6C structures, respectively. The distance between OT1(Ile839) and the ligated water O was 2.52 Å for both the 5C and 6C geometries. The angle formed by OT1(Ile839), the ligated water H, and the ligated water O was 165° for both the 5C and 6C structures.