The Structural Origin of Nonplanar Heme Distortions in Tetraheme Ferricytochromes c 3 ² Jian-Guo Ma, ‡ Jun Zhang, ‡ Ricardo Franco, § Song-Ling Jia, ‡ Isabel Moura, § Jose ´ J. G. Moura, § Peter M. H. Kroneck, | and John A. Shelnutt* ,‡ Materials Theory and Computation Department, Sandia National Laboratories, Albuquerque, New Mexico 87185-1349, Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 87131, Centro de Quı ´mica Fina e Biotecnologia, Departamento de Quı ´mica, Faculdade de Cie ˆ ncias e Tecnologia, UniVersidade NoVa de Lisboa, 2825 Monte de Caparica, Portugal, and Fakulta ¨ t fu ¨ r Biologie, UniVersita ¨ t Konstanz, D-78457 Konstanz, Germany ReceiVed May 20, 1998; ReVised Manuscript ReceiVed July 8, 1998 ABSTRACT: Resonance Raman (RR) spectroscopy, molecular mechanics (MM) calculations, and normal- coordinate structural decomposition (NSD) have been used to investigate the conformational differences in the hemes in ferricytochromes c 3 . NSD analyses of heme structures obtained from X-ray crystallography and MM calculations of heme-peptide fragments of the cytochromes c 3 indicate that the nonplanarity of the hemes is largely controlled by a fingerprint peptide segment consisting of two heme-linked cysteines, the amino acids between the cysteines, and the proximal histidine ligand. Additional interactions between the heme and the distal histidine ligand and between the heme propionates and the protein also influence the heme conformation, but to a lesser extent than the fingerprint peptide segment. In addition, factors that influence the folding pattern of the fingerprint peptide segment may have an effect on the heme conformation. Large heme structural differences between the baculatum cytochromes c 3 and the other proteins are uncovered by the NSD procedure [Jentzen, W., Ma, J.-G., and Shelnutt, J. A. (1998) Biophys. J. 74, 753-763]. These heme differences are mainly associated with the deletion of two residues in the covalently linked segment of hemes 4 for the baculatum proteins. Furthermore, some of these structural differences are reflected in the RR spectra. For example, the frequencies of the structure-sensitive lines (ν 4 , ν 3 , and ν 2 ) in the high-frequency region of the RR spectra are lower for the Desulfomicrobium baculatum cytochromes c 3 (Norway 4 and 9974) than for the DesulfoVibrio (D.) gigas, D. Vulgaris, and D. desulfuricans strains, consistent with a more ruffled heme. Spectral decompositions of the ν 3 and ν 10 lines allow the assignment of the sublines to individual hemes and show that ruffling, not saddling, is the dominant factor influencing the frequencies of the structure-sensitive Raman lines. The distinctive spectra of the baculatum strains investigated are a consequence of hemes 2 and 4 being more ruffled than is typical of the other proteins. Cytochrome c 3 is a tetraheme protein found in sulfate- reducing bacteria, which are capable of using sulfate of thio- sulfates as the terminal electron acceptor. X-ray crystal structures of cytochromes c 3 from the organisms De- sulfoVibrio gigas (1), D. 1 Vulgaris [Hildenborough (2, 3) and Miyazaki (4)], D. desulfuricans (5), and Desulfomicrobium baculatum (6, 7) have been solved, showing that the general outlines of their protein structure are essentially the same. Specifically, the X-ray structures indicate that the overall protein folding is conserved (5). The structural similarity occurs although the amino acid sequences exhibit as little as about 20% identity, and this mostly resulting from the eight conserved histidine axial ligands and eight cysteine residues linked to the hemes. A recent analysis of the crystal structures provides a more detailed understanding of the structures of the hemes in these proteins (8). It was found that the conformations of the four hemes are different from each other, but the structures are conserved for corresponding hemes. The exception is hemes 4, which exhibits a very different structure for Dsm. baculatum cytochromes c 3 . In the present work, we explore the structural origin of the heme distortions in these proteins. Nonplanar distortions of tetrapyrroles are prevalent in the hemes of hemoproteins, the pigments of photosynthetic proteins, and cofactor F 430 of methylreductase (9). The nonplanarity of these porphyrin cofactors is believed to influence the biological activity of the proteins, in part, because the porphyrin deformations are often conserved within functional classes of proteins (8, 9). The hemopro- teins provide a representative example of the occurrence of nonplanar porphyrins in proteins. It has been recognized for about 10 years that the hemes in many hemoproteins are highly distorted from planarity and that these nonplanar ² Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-AC04-94DP85000. I.M., J.J.G.M., and R.F. want to thank the PRAXIS Program for financial support. * To whom correspondence should be addressed. ‡ Sandia National Laboratories and University of New Mexico. § Universidade Nova de Lisboa. | Universita ¨t Konstanz. 1 Abbreviations: RR, resonance Raman; MM, molecular mechanics; NSD, normal-coordinate structural decomposition; MP, microperoxi- dase; D., DesulfoVibrio; Dsm., Desulfomicrobium. 12431 Biochemistry 1998, 37, 12431-12442 S0006-2960(98)01189-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/20/1998