Structural Characterization of Synthetic and Protein-Bound Porphyrins in Terms of the Lowest-Frequency Normal Coordinates of the Macrocycle

Abstract

The X-ray crystal structures of synthetic and protein-bound metalloporphyrins are analyzed using a new normal structural decomposition method for classifying and quantifying their out-of-plane and in-plane distortions. These distortions are characterized in terms of equivalent displacements along the normal coordinates of the D 4 h -symmetric porphyrin macrocycle (normal deformations) by using a computational procedure developed for this purpose. Often it turns out that the macrocyclic structure is, even in highly distorted porphyrins, accurately represented by displacements along only the lowest-frequency normal coordinates. Accordingly, the macrocyclic structure obtained from just the out-of-plane normal deformations of the saddling ( sad, B 2u )-, ruffling ( ruf, B 1u )-, doming ( dom, A 2u )-, waving [ wav ( x ), wav ( y ); E g ]-, and propellering ( pro, A 1u )-type essentially simulates the out-of-plane distortion of the X-ray crystal structure. Similarly, the observed in-plane distortions are decomposed into in-plane normal deformations corresponding to the lowest-frequency vibrational modes including macrocycle stretching in the direction of the meso -carbon atoms ( meso-str, B 2g ), stretching in the direction of the nitrogen atoms ( N-str, B 1g ), x and y pyrrole translations [ trn ( x ), trn ( y ); E u ], macrocycle breathing ( bre, A 1g ), and pyrrole rotation ( rot, A 2g ). The finding that the displacements of the 24 atoms of the macrocycle primarily occur along the lowest-frequency normal coordinates is expected on physical grounds and is verified by structural decomposition of more than 100 synthetic and 150 protein-bound metalloporphyrin X-ray crystal structures. Because of the high resolution of the X-ray crystal structures of synthetic metalloporphyrins, the small displacements for other normal coordinates are also able to be discerned. However, for the heme groups in proteins, only the displacements along the lowest-frequency modes are detectable because of the large uncertainties in the atomic positions. The heme groups in the four X-ray crystal structures of deoxyhemoglobin are used to evaluate the structural decomposition method. We find that the corresponding heme groups in different X-ray crystal structures are similar. Furthermore, the out-of-plane distortions for the heme groups in the α- and β-chains are found to be inequivalent, that is, the two α-heme groups are mainly ruffled and domed whereas the two β-heme groups are primarily saddled and domed. In the case of isozyme-1 ferrocytochrome c and its mutants, the heme distortion is not significantly influenced by the point mutations, and the strongly nonplanar structure is most likely the result of interactions of the heme group with a small protein segment, probably the linkage Cys−Leu−Gln−Cys−His. This conclusion is in agreement with previous findings that the heme distortion in cytochrome c is conserved for the proteins for which X-ray crystal structures exist and significant structural variation occurs only when an amino acid difference appears in Cys−X−Y−Cys−His segment. A similar conclusion is suggested by the structural decomposition results of the four heme groups in cytochromes c 3 . The analogous covalently linked peptide segments vary for each heme group, giving different distortions for the four hemes. Nevertheless, the distinctive distortion of each heme group is conserved for cytochromes c 3 from different species as long as the short peptide sequences between the cysteine linkages are homologous.