Electrochemical and NMR spectroscopic studies of distal pocket mutants of nitrophorin 2: Stability, structure, and dynamics of axial ligand complexes

WT and leucine --> valine distal pocket mutants of nitrophorin 2 (NP2) and their NO complexes have been investigated by spectroelectrochemistry. NO complexes of two of the mutants exhibit more positive reduction potential shifts than does the WT protein, thus indicating stabilization of the Fe(II)-NO state. This more positive reduction potential for NP2-L132V and the double mutant is consistent with the hypothesis that smaller valine residues may allow the heme to regain planarity instead of being significantly ruffled, as in WT NP2. Thus, ruffling may stabilize the Fe(III)-NO state, which is required for facile NO dissociation. NMR spectroscopic investigations show that the sterically demanding 2-methylimidazole ligand readily binds to all three distal pocket mutants to create low-spin Fe(III) complexes having axial ligands in nearly perpendicular planes; it also binds to the WT protein in the presence of higher concentrations of 2-methylimidazole, but yields a different ligand plane orientation than is present in any of the three distal pocket mutants. NOESY spectra of NP2-ImH mutants exhibit chemical exchange cross peaks, whereas WT NP2-ImH shows no chemical exchange. Chemical exchange in the case of the distal leucine --> valine mutants is caused by ImH ligand orientational dynamics. The two angular orientations of the ImH ligand could be determined from the H-1 chemical shifts of the heme methyls, and the rate of interconversion of the two forms could be estimated from the NOESY diagonal and cross peak intensities. K-eq is 100 or larger and favors an orientation similar to that found for the WT NP2-ImH complex.