Mitochondrial Fe-S cluster bioassembly pathway
Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and pathway signaling. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to activate Nfs1, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as the Fe(II) chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. The goal of the Stemmler laboratory is to provide molecular and atomic level detail of the structure and function of all the ISC proteins, within the yeast and fly model systems, alone and in complex with all protein partners. Four articles that highlight our contribution to this field include:
- Rodrigues, A.V.*; Kandegedara, A.*; Rotondo, J.A.*; Dancis, A.; Stemmler, T.L. :Iron Loading Site on the Fe-S Cluster Assembly Scaffold Protein is Distinct from the Active Site” BioMetals, 2015, In Press.
- Pandey, A.; Gordon, D.M.; Pain, J.; Stemmler, T.L.; Dancis, A.; Pain, D. “Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.” J. Biol. Chem., 2013, 288, 36773-86. PMID: 24217246.
- Cook, J.D.*; Kondapalli, K.C.*; Rawat, S.*; Childs, W.C.*; Murugesan, Y.*; Dancis, A.; Stemmler, T.L. “Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly” Biochem., 2010, 49 (3), 8756-65. PMID: 20815377
- Kondapalli, K.C.*; Kok, N.M.*; Dancis, A.; Stemmler, T.L. “Drosophila Frataxin: an iron chaperone during cellular [2Fe-2S] cluster bioassembly” Biochem., 2008, 47, 6917-27. PMID: 18540637
Structure and function of the poly (rC) binding protein (PCBP) family
Cellular pathways directing metal storage and utilization are essential for preventing iron toxicity during overload and ensuring availability during deficiency. Storage is directed by the 4-helix bundle protein ferritin while metal utilization is a shared function by several proteins within the cytosol and across cellular compartments. Recently, the human poly (rC)-binding protein (PCBP) was shown to function as a cytosolic iron chaperone that delivers Fe2+ to ferritin and to several other protein partners. Mammalian cytoplasmic ferritins exist as a 24 subunits multimer of 2 ferritin subunits: H chain (21,000 kDa), which controls the ferroxidase chemistry coupled to iron storage, and the L chain (19,000 kDa). Apo-ferritin H and L chains self-assemble to form a spherical protein shell that stores up to 4500 iron atoms as feroxyhydrite within the central cavity. PCBP is essential for delivery of metal to ferritin suggesting a direct role of metal delivery to the protein complex. In addition, PCBP has been shown to serve as an iron chaperone for delivery of metal loaded into the active site of several metalloproteins. The goal of the Stemmler lab is to provide a molecular level characterization of the PCBP protein family and elucidate their mechanisms for iron delivery. Four articles that highlight our contribution to this field include:
- Leidgens, S.; Bullough, K.Z.; Shi, H.; Shakoury-Elizeh, M.; Yabe, T.; Subramanian, P.*; Hsu, E.; Natarajan, N.; Nandal, A.; Stemmler, T.L.; Philpott, C.C. “Each member of the PCBP family exhibits iron chaperone activity towards ferritin” J. Biol. Chem., 2013, 228, 17791-802. PMID: 23640898.
- Nandal, A.; Ruiz, J.C.; Subramanian, P.*; Ghimire-Rijal, S.*; Sinnamon, R.A.*; Stemmler, T.L.; Bruick, R.K.; Philpott, C.C. “Activation of the HIF Prolyl Hydroxylase by the Iron Chaperones PCBP1 and PCBP2” Cell Metab., 2011, 14 (5), 647-57. PMID: 22055506.
- Subramanian, P.*; Rodrigues, A.*; Ghimire, S.R.*; Stemmler, T.L. “Iron chaperones for mitochondrial Fe-S cluster biosynthesis and ferritin iron storage” Cur. Opin. Chem. Biol., 2011, 15, 312-8. PMID: 21288761.
- Shi, H.; Bencze, K.Z.*; Stemmler, T.L.; Philpott, C.C. “A cytosolic iron chaperone that delivers iron to ferritin” Science, 2008, 320, 1207-10. PMID: 18511687.
Characterization of the metal centers in particulate methane monooxygenase
Methanotrophic bacteria are organisms that oxidize methane to methanol in the first step of their metabolism. These proteins have been increasingly shown to be important in the quest for efficient conversion of abundant natural gas to useable fuels and chemicals. Methane oxidation is catalyzed by the methane monooxygenase enzymes. Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol within methanotrophic bacteria. pMMO is composed of three subunits: pmoB, pmoA, and pmoC arranged in an 33c3 trimer. The pmoB subunit comprises two periplasmic domains connected by two transmembrane helices. In Methylococcus capsulatus (Bath) pMMO, these soluble domains house two copper sites, modeled as monocopper and dicopper. The goal of the Stemmler lab has been to characterize the structure and electronic properties of the pMMO copper centers, working in collaborations with Dr. Amy Rosenzweig’s laboratory. Four articles that highlight our contribution to this field include:
- Sirajuddin, S.; Barupala, D.*; Helling, S.; Marcus, K.; Stemmler, T.L.; Rosenzweig, A.C. “Effects of Zinc on Particulate Methane Monooxygenase Activity and Structure” J Biol Chem, 2014, 289, 21782-94. PMID: 24942740.
- Smith, S.M.; Rawat, S.*; Telser, J.; Hoffman, B.M.; Stemmler, T.L.; Rosenzweig, A.C. “Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M” Biochem., 2011, 59 (1), 10231-40. PMID: 22013879.
- Balasubramanian, R.; Smith, S.M.; Rawat, S.*; Yatsunyk, L.A.; Stemmler, T.L.; Rosenzweig, A.C. “Oxidation of methane by a biological dicopper center” Nature, 2010, 465, 115-9. PMID: 20410881.
- Hakemian, A.S.; Kondapalli, K.C.*; Telser, J; Hoffman, B.M.; Stemmler, T.L.; Rosenzweig, A.C. “The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b” Biochem., 2008, 47, 6793-801. PMID: 18540635.
Cellular transport and processing of metals
The P-type ATPases are a family of integral membrane proteins that use energy from ATP hydrolysis to transport cations across cell membranes. The P1B-type ATPases are the most widely distributed group within this ATPase superfamily and these transmembrane proteins confer diverse heavy metal tolerance to microorganisms. They are characterized by a shared core architecture, consisting of six to eight transmembrane helices, a soluble cytosolic ATP binding domain present between the second and third-to-last transmembrane regions and a preceding cytosolic actuator domain. The P1B-ATPases are responsible for transition metal efflux out of the cytosol into either the extracellular space or into the lumen of intracellular organelles. Since the mechanism for transport and factors that dictate metal specificity for the P1B-ATPases are unknown, the goal of the Stemmler lab is through collaboration with the Rosenzweig, Argüello and Rosen laboratories to characterize aspects of metal association and utilization by this important class of proteins. Four articles that highlight our contribution to this field include:
- Zielazinski, E.L.: González-Guerrero, M.; Subramanian, P.*; Stemmler, T.L.; Argüello, J.M.; Rosenzweig, A.C. “Sinorhizobium meliloti Nia is a P(1B-5)-ATPase expressed in the nodule during plant symbiosis and is involved in Ni and Fe transport.” Metallomics, 2013, 12, 1614-23. PMID: 22971227.
- Zielazinski, E.L.; Cutsail III, G.E.; Hoffman, B.M.; Stemmler, T.L.; Rosenzweig, A.C. “Characterization of a Cobalt-Specific P1B-ATPase” Biochem., 2012, 51 (40), 7891-900. PMID: 22971227.
- Raimunda, D.; Subramanian, P.*; Stemmler, T.; Argüello, J.M. “A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn(2+)-ATPases” Biochem. Biophys. Acta., 2012, 1818 (5), 1374-7. PMID: 22387457.
- Traverso, M.E.; Subramanian, P.*; Davydov, R.; Hoffman, B.M.; Stemmler, T.L.; Rosenzweig, A.C. “Identification of a hemerythrin-like domain in a P1B-type transport ATPase” Biochem., 2010, 49 (33), 7060-8. PMID: 20672819.
Cellular metal storage and utilization
Inorganic elements are central to life’s processes because they enable catalysis of reactions that are not readily enabled by the functional groups found on the side chains of amino acids. This includes zinc, which serves as an electrophile in numerous reactions, as well as copper, iron and manganese, which serve as cofactors to enable redox chemistry critical for extracting energy from oxidation of inorganic compounds for autotrophic growth or organic compounds for heterotrophic growth. Given the reactivity of these transition metals, cells ensure the chemistry associated with each metal is controlled through pathways that direct metal homeostasis and ensure metals are loaded into appropriate recipient proteins. As an example, Cu homeostasis in C. reinhardtii is disrupted by nutritional Zn deficiency, which results in unprecedented Cu accumulation of up to 20 times the typical quota. In collaboration with the Merchant laboratory, we have characterized the structural and redox properties of Fe, Cu and Zn storage within C. reinhardtii cells, with the goal of characterizing pathways that leads to metal accumulation. An article that highlights our contribution to this field include:
- Hong-Hermesdorf, A.; Miethke, M.; Gallaher, S.D.; Kropat,J.; Dodani, S.C.; Barupala, D.*; Chan, J.; Domaille, D.W.; Shirasaki, D.I.; Loo, J.A.; Weber, P.K.; Pett-Ridge, J.; Stemmler, T.L.; Chang, C.J.; Merchant, S.S. “Selective sub-cellular visualization of trace metals identifies dynamic sites of Cu accumulation in Chlamydomonas” Nature Chemical Biology, 2014, 10, 1034-42. PMID: 25344811.