Insight into the function of active site residues in the catalytic mechanism of human ferrochelatase.

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase.

Heme is an iron-containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was performed to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK 293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in the level of hemoglobinization relative to that of untreated cells. In vitro heme transfer experiments showed that purified PGRMC1 was able to donate heme to apo-cytochrome b5. In the presence of PGRMC1, in vitro measured FECH activity decreased in a dose-dependent manner. Interactions between FECH and PGRMC1 were strongest for the conformation of FECH associated with product release, suggesting that PGRMC1 may regulate FECH activity by controlling heme release. Overall, the data illustrate a role for PGRMC1 in regulating heme synthesis via interactions with FECH and suggest that PGRMC1 may be a heme chaperone or sensor.

Identification of the Mitochondrial Heme Metabolism Complex.

Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.