Prediction of reduction potential changes in rubredoxin: a molecular mechanics approach

Abstract

Predicting the effects of mutation on the reduction potential of proteins is crucial in understanding how reduction potentials are modulated by the protein environment. Previously, we proposed that an alanine vs. a valine at residue 44 leads to a 50-mV difference in reduction potential found in homologous rubredoxins because of a shift in the polar backbone relative to the iron site due to the different side-chain sizes. Here, the aim is to determine the effects of mutations to glycine, isoleucine, and leucine at residue 44 on the structure and reduction potential of rubredoxin, and if the effects are proportional to side-chain size. Crystal structure analysis, molecular mechanics simulations, and experimental reduction potentials of wild-type and mutant Clostridium pasteurianum rubredoxin, along with sequence analysis of homologous rubredoxins, indicate that the backbone position relative to the redox site as well as solvent penetration near the redox site are both structural determinants of the reduction potential, although not proportionally to side-chain size. Thus, protein interactions are too complex to be predicted by simple relationships, indicating the utility of molecular mechanics methods in understanding them.

FIGURE 1

A ribbon diagram of Cp (1IRO) rubredoxin crystal structure is shown with β-strands in blue and backbone in gray. The iron (red) and the sulfurs (yellow) of the redox site and the side chains of residue 8 (green), 41 (purple), and 44 (blue) are also shown in a ball-and-stick model. This figure was generated using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1994).

FIGURE 2

A licorice model of the side chain of leucine 41 in both the open (purple) and closed conformation (translucent purple) reported in the crystal structure; the iron (red) and the sulfurs (yellow) of the redox site and the backbone of the protein (gray) are also shown. This figure was generated using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1994).

FIGURE 3

A licorice model of the side-chain interaction of residue 44 and residue 8 for the WT (gray), Val44Ala (purple), and Val44Gly (magenta) and the iron (red) and the sulfurs (yellow) of the redox site in the crystal structures with the polar hydrogen atoms built in, and the van der Waals surfaces of WT represented with transparent spheres. The orientation of the peptide dipole of the WT protein is indicated by the red (−) to blue (+) arrow. This figure was generated using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1994).

FIGURE 4

A licorice model depicting the backbone conformations for the WT crystal structure (gray) and two snapshots from the MD simulation of the Val44Gly mutant in the φ ≈ −75° conformation (magenta) and the φ ≈ −160° conformation (pink); the iron (red) and the sulfurs (yellow) of the redox site are also shown. The change in the peptide dipole orientation between the two conformations is shown with a double-headed arrow pointing toward the respective H₄₄. This figure was generated using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1994).

FIGURE 5

A licorice model depicting the side-chain interaction of residue 44 and residue 8 for the WT (gray), Val44Ile (blue), Val44Leu (green), and the iron (red) and the sulfurs (yellow) of the redox site in the EM structures with the van der Waals surfaces of WT represented with transparent spheres. The orientation of the peptide dipole of the WT protein is indicated by the red (−) to blue (+) arrow. This figure was generated using MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1994).