Assignment of the ferriheme resonances of the high-spin forms of nitrophorins 1 and 4 by 1H NMR spectroscopy: comparison to structural data obtained from X-ray crystallography.

In this work, we report the assignment of the majority of the ferriheme resonances of high-spin nitrophorins (NPs) 1 and 4 and compare them to those of NP2, published previously. It is found that the structures of the ferriheme complexes of NP1 and NP4, in terms of the orientation of the histidine imidazole ligand, can be described with good accuracy by NMR techniques and that the angle plot proposed previously for the high-spin form of the NPs (Shokhireva, T. Kh.; Shokhirev, N. V.; Walker, F. A. Biochemistry 2003, 42, 679-693) describes the angle of the effective nodal plane of the axial histidine imidazole in solution. There is an equilibrium between the two heme orientations (A and B), which depends on the heme cavity shape, which can be altered by mutation of amino acids with side chains (phenyl vs tyrosyl) near the potential position where a heme vinyl group would be in one of the isomers. The A:B ratio can be much more accurately measured by NMR spectroscopy than by X-ray crystallography.

Assignment of the ferriheme resonances of the low-spin complexes of nitrophorins 1 and 4 by (1)H and (13)C NMR spectroscopy: comparison to structural data obtained from X-ray crystallography.

In this work we report the assignment of the majority of the ferriheme resonances of low-spin nitrophorins (NP) 1 and 4 and compare them to those of NP2, published previously. It is found that the structure of the ferriheme complexes of NP1 and NP4, in terms of the orientation of the ligand(s), can be determined with good accuracy by NMR techniques in the low-spin forms and that angle plots proposed previously (Shokhirev, N. V.; Walker, F. A. J. Biol. Inorg. Chem. 1998, 3, 581-594) describe the angle of the effective nodal plane of the axial ligands in solution. The effective nodal plane of low-spin NP1, NP4, and NP2 complexes is in all cases of imidazole and histamine complexes quite similar to the average of the His-59 or -57 and the exogenous ligand angles seen in the X-ray crystal structures. For the cyanide complexes of the nitrophorins, however, the effective nodal plane of the axial ligand does not coincide with the actual histidine-imidazole plane orientation. This appears to be a result of the contribution of an additional source of asymmetry, the orientation of one of the zero-ruffling lines of the heme. Probably this effect exists for the imidazole and histamine complexes as well, but because the effect of asymmetry that occurs from planar exogenous axial ligands is much larger than the effect of heme ruffling the effect of the zero-ruffling line can only be detected for the cyanide complexes, where the only ligand plane is that of the proximal histidine. The three-dimensional structures of the three NP-CN complexes, including that of NP2-CN reported herein, confirm the high degree of ruffling of these complexes. There is an equilibrium between the two heme orientations (A and B) that depends on the heme cavity shape and changes somewhat with exogenous axial ligand. The A:B ratio can be much more accurately measured by NMR spectroscopy than by X-ray crystallography.

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 (1)H 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.