The Conserved Active-Site Loop Residues of Ferrochelatase Induce Porphyrin Conformational Changes Necessary for Catalysis ² Zhen Shi, ‡ Ricardo Franco, § Raid Haddad, |,⊥ John A. Shelnutt,* ,⊥,@ and Gloria C. Ferreira* ,‡,# Department of Biochemistry and Molecular Biology, College of Medicine, and H. Lee Moffitt Cancer Center and Research Institute, UniVersity of South Florida, Tampa, Florida 33612-4799, REQUIMTE, Departamento de Quı ´mica, Faculdade de Cie ˆ ncias e Tecnologia, UniVersidade NoVa de Lisboa, 2829-516 Caparica, Portugal, Department of Chemical and Nuclear Engineering, UniVersity of New Mexico, Albuquerque, New Mexico 87131, Surface and Interface Sciences Department, Sandia National Laboratories, Albuquerque, New Mexico 87185-1349, and Department of Chemistry, UniVersity of Georgia, Athens, Georgia 30602-2556 ReceiVed September 19, 2005; ReVised Manuscript ReceiVed December 7, 2005 ABSTRACT: Binding of porphyrin to murine ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is investigated by employing a set of variants harboring mutations in a putative porphyrin- binding loop. Using resonance Raman (RR) spectroscopy, the structural properties of the ferrochelatase- bound porphyrins are examined, especially with respect to the porphyrin deformation occurring in the environment of the active site. This deformation is thought to be a key step in the enzymatic insertion of ferrous iron into the porphyrin ring to make heme. Our previous RR spectroscopic studies of binding of porphyrin to murine ferrochelatase led us to propose that the wild-type enzyme induces porphyrin distortion even in the absence of the metal ion substrate. Here, we broaden this view by presenting evidence that the degree of a specific nonplanar porphyrin deformation contributes to the catalytic efficiency of ferrochelatase and its variants. The results also suggest that the conserved Trp256 (murine ferrochelatase numbering) is partially responsible for the observed porphyrin deformation. Binding of porphyrin to the ferrochelatase variants causes a decrease in the intensity of RR out-of-plane vibrational mode γ 15 , a saddling- like mode that is strong in the wild-type enzyme. In particular, the variant with a catalytic efficiency 1 order of magnitude lower than that of the wild-type enzyme is estimated to produce less than 30% of the wild-type saddling deformation. These results suggest that specific conserved loop residues (especially Trp256) are directly involved in the saddling of the porphyrin substrate. The terminal step in heme biosynthesis is catalyzed by ferrochelatase (EC 4.99.1.1, protoheme ferrolyase), which inserts ferrous iron into protoporphyrin IX to produce protoheme (1-3). Identified in a large number of organisms, ferrochelatase exhibits a folding pattern and an active-site structure with a high degree of homology across taxa (1, 2). As shown in the X-ray crystal structures of the enzyme from Bacillus subtilis (4, 5), human (6), and Saccharomyces cereVisiae (7), the monomeric unit consists of two domains, each with a Rossmann-type fold. Porphyrin binds to a deep cleft between the two domains (5), which is rich in conserved residues proposed to be important in metal and porphyrin binding and catalysis (7, 8). The most widely accepted reaction mechanism for ferro- chelatase suggests that a critical step involves distortion of the porphyrin macrocycle which exposes the lone-pair orbitals of the pyrrole nitrogens to the incoming metal ion (9). This notion has received support from both experimental and theoretical studies (5, 10-12) which show that an out- of-plane distortion occurs upon substrate binding. The X-ray structure of the strong complex between bacterial ferroche- latase and the substrate analogue N-methylmesoporphyrin showed deformation of the macrocycle, including tilting of pyrrole ring A and distortion of the other pyrroles to yield a predominantly saddled-ruffled structure (5). Although this porphyrin conformation would necessarily result from the N-methyl substitution, the high affinity of the protein for porphyrin locked into this conformation suggests that it corresponds to a preferred porphyrin structure in the active site. Moreover, antibodies raised against nonplanar alkylated porphyrins were found to catalyze porphyrin metalation by Zn 2+ and Cu 2+ (12). Resonance Raman (RR) 1 spectra of the antibody-bound mesoporphyrin revealed an out-of-plane distortion (10), and the X-ray structure of the Michaelis ² This work was supported by American Cancer Society Grant RSG- 96-05106-TBE to G.C.F. Sandia National Laboratories is a multipro- gram laboratory operated by Sandia Corp., a Lockheed Martin company, for the U.S. Department of Energy under Contract DE-AC04- 94AL85000. * To whom correspondence should be addressed. G.C.F.: Depart- ment of Biochemistry and Molecular Biology, College of Medicine, MDC 7, University of South Florida, Tampa, FL 33612-4799; telephone, (813) 974-5797; fax, (813) 974-0504; e-mail, gferreir@ hsc.usf.edu. J.A.S.: Sandia National Laboratories, Albuquerque, NM 87185-1349; telephone, (505) 272-7160; fax, (505) 272-7077; e-mail, jasheln@unm.edu. ‡ Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida. § Universidade Nova de Lisboa. | University of New Mexico. ⊥ Sandia National Laboratories, Albuquerque. @ University of Georgia. # H. Lee Moffitt Cancer Center and Research Institute, University of South Florida. 2904 Biochemistry 2006, 45, 2904-2912 10.1021/bi051907i CCC: $33.50 © 2006 American Chemical Society Published on Web 02/07/2006