Evidence That the Fourth Ligand to the [2Fe-2S] Cluster in Animal Ferrochelatase Is a Cysteine

Abstract

The terminal enzyme in heme biosynthesis, ferrochelatase (E.C. 4.99.1.1), catalyzes the insertion of iron into protoporphyrin IX. Nuclear-encoded and produced in the cytoplasm, ferrochelatase is proteolytically processed upon translocation into the mitochondrion. In eukaryotes, the mature-length 42,000 Da protein is associated with the inner mitochondrial membrane, with the active site facing the mitochondrial matrix (1). The proposed catalytic mechanism (2) initially involves a metaldependent, enzyme-mediated distortion of the bound porphyrin ring allowing rapid insertion of Fe 2ϩ into the bent porphyrin (3). Distinct from the enzyme's catalytic activity, a labile 2ϩ cluster has been identified in several animal ferrochelatases including human (4), mouse (5), chicken, and frog (6). Although similar to the [2Fe-2S] 2ϩ centers found in plant ferredoxins, the ferrochelatase iron-sulfur cluster is more labile with enhanced sensitivity to degradation by nitric oxide (7). That the cluster plays no direct role part in catalysis is evidenced by the observation that bacterial, plant, and yeast ferrochelatases do not contain the metal center, and by studies showing that the redox state of the cluster is inconsequential to enzyme activity (4). However, when the cluster is disassembled, enzyme activity is lost (4, 7) and the protein readily precipitates. Hence the cluster appears to play a crucial role in maintaining protein structure in animal ferrochelatases and coupled with the sensitivity to nitric oxide, this has lead us to postulate that the cluster may serve as a regulatory NO 1 -sensor as part of an immune response (7).Site-directed mutagenesis of the five conserved cysteines closest to the COOH terminus of recombinant human ferrochelatase, identified Cys-403, Cys-406, and Cys-411 as ligands to the [2Fe-2S] cluster (8). Additionally, at this time it was proposed that certain spectroscopic anomalies and the unusual lability of the cluster may result from one noncysteinyl oxygenic ligand. The objective of the present investigation was to identify the fourth cluster ligand via additional mutagenesis experiments and by cloning, expression, and characterization of cluster-containing ferrochelatases from more distantly related organisms. We report here the first characterization of ferrochelatase from Drosophila melanogaster and, together with the new mutagenesis results with the human enzyme, these new data support the assignment of the cysteine located at position 196 in the human ferrochelatase as the fourth cluster ligand. Tracing the biological evolution of the ironsulfur cluster in ferrochelatase also provides further insight concerning the specific role of this metal center. EXPERIMENTAL PROCEDURESStrains and Cell Culture-Escherichia coli strain JM109 was used to express recombinant human ferrochelatase, mutant human ferrochelatase, and recombinant Drosophila ferrochelatase as described elsewhere (9). For the mutagenesis procedure described below, E. coli strain BMH mut-s was used to amplify the mutant plasmid. E...

Posttranslational stability of the heme biosynthetic enzyme ferrochelatase is dependent on iron availability and intact iron-sulfur cluster assembly machinery

“…14 The crystal structure of human ferrochelatase suggests a structural role for the Fe-S cluster by revealing a unique stabilizing bridge formed by the cluster between the 3 carboxy-terminal cysteines and a fourth internal cysteine. 12,15 Mutations in the 4 coordinating cysteine residues of recombinant human ferrochelatase resulted in an inactive enzyme, 15 . Initial demonstration of decreased ferrochelatase activity in cultured rat hepatocytes treated with S-nitroso-N-acetylpenicillamine, 19 a nitric oxide (NO)-generating compound, prompted 2 further studies, which showed that the cluster is sensitive to destruction by NO in vitro, 20 and in cultured cells.…”

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

“…The two domains associate to form a cleft, in which conserved residues, proposed to be critical in the binding of substrates and in the proper functioning of the enzyme, are located [5]. In contrast to the bacterial and yeast ferrochelatases, which appear to be devoid of cofactors, vertebrate and Drosophila melanogaster ferrochelatases are metalloenzymes with a [2Fe-2S] cluster [7][8][9][10]. Despite having no direct catalytic role, the iron-sulphur cluster is essential for activity of the enzyme [11].…”

An Intestinal Parasitic Protist, Entamoeba histolytica, Possesses a Non-redundant Nitrogen Fixation-like System for Iron-Sulfur Cluster Assembly under Anaerobic Conditions

“…Despite common catalytic and scaffold mechanisms shared by the NIF, ISC, and SUF systems, there are a number of differences among these systems (10); the ISC and SUF systems appear to be considerably more complex than the NIF system. For example, heat-shock-cognate (Hsc) proteins have been suggested to have chaperone-like functions in the ISC system for the formation of transient Fe-S clusters or insertion into various target proteins in bacteria and yeast (7,26,30,50,51). Our in vivo complementation study revealed that the NIF-like system does not require any additional component other than NifS and NifU for Fe-S cluster assembly under anaerobic conditions.…”