 |
Danilo Ortillo , Post-doctoral Research AssociateContact Information:Montana State UniversityDepartment of Chemistry and Biochemistry226 Chemistry and Biochemistry & BuildingBozeman, MT 59717
|
Research Interests
Biological Fe-S Clusters
Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. Owing to their remarkable structural plasticity and versatile chemical/electronic features, [Fe-S] clusters participate in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity1.1. Johnson, D. C.; Dean, D.R.; Smith, A. D.; Johnson, M. K., Annu. Rev. Biochem, 2005, 74, 247
Hydrogenases
Hydrogen metabolism is facilitated by the activity of three evolutionary and structurally unrelated enzymes: the [Ni-Fe]-hydrogenases, [Fe-Fe]-hydrogenases and [Fe]-hydrogenases2,3. The catalytic core of the [Fe-Fe]-hydrogenase (HydA), termed the H-cluster (Figure 1), is bound to the protein by four cysteine ligands of the [4Fe-4S] subcluster, which is further linked by a cysteine thiolate ligand to a 2Fe subcluster with unique non-protein ligands containing CO, CN- and a dithiolate ligand. A water molecule is coordinated to the distal Fe of the 2Fe subcluster in the presumed-active oxidized state4. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF, and HydG; however the mechanism, synthesis and means of insertion of the H-cluster components remain unclear5.2. Vignais, P.M.; Billoud, B., Chem. Rev., 2007, 107, 4206.
3. Shima, S.; Thauer, R.K., Chem. Rev., 2007, 7, 37.
4. Panday, A.S.; Harris, T.V.; Giles, L. J.; Peters, J.W.; Szilagyi, R. K.; J. Am. Chem. Soc., 2008, 130, 4533.
5. Mulder, D.; Ortillo, D.O.; Gardenghi, D.J.; Naumov, A.V.; Ruebush, S.S.; Szilagyi, R.K.; Huynh, B.; Broderick, J.B.; Peters, J.W., Biochemistry,
2009, 48, 6240.
Mӧssbauer Spectroscopy of HydAΔEFG
Purified protein of HydDEFG exhibits UV-visible spectroscopic features characteristic of Fe-S clusters. The reduced protein gave rise to an axial S=1/2 EPR signal (g=2.04 and 1.91) characteristic of a reduced [4Fe-4S]+ cluster. Mӧssbauer spectroscopic characterization of 57Fe enriched HydAΔEFG (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) provided further evidence of the presence of a redox active [4Fe-4S]2+/+ cluster. As-purified HydAΔEFG (A) show that majority of the Fe is present in the oxidized [4Fe4S]2+ form while a small percentage is in the reduced [4Fe-4S]+ form. The remaining 15% is present as FeII impurities. (Figure 2) Upon reduction (B and C), a substantial amount of the [4Fe4S]2+clusters are reduced to the [4Fe-4S]+ form5.
[Fe-Fe]-hydrogenase H-cluster Assembly
The structure of HydAΔEFG reveals the presence of the [4Fe-4S] cluster and an open pocket for the 2Fe subcluster. This indicates that the synthesis of the H-cluster occurs in a stepwise manner, first with synthesis and insertion of the [4Fe-4S] subcluster by the generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery6.6. Mulder, D.W.; Boyd, E.S.; Sarma, R.; Lange, R.K.; Endrizzi, J.A.; Broderick, J.B.; Peters,J.W., Nature, 2010, 465, 248.
NfUA
NifU is a modular protein that contains three distinct domains with the central domain containing a stable redox active [2Fe-2S] cluster with an as-yet-unknown function. In vitro and in vivo experiments have established that labile [Fe-S] clusters can be assembled on both the N-terminal and C-terminal domains of NifU, and such cluster-loaded forms of NifU can be used for activation of the nitrogenase Fe-protein. Located elsewhere on the A. vinelandii genome is a gene, designated as nfuA, whose product encodes a protein having a C-terminal sequence similar to the C-terminal domain of NifU (Figure 4). The sequence conservation between NifU and NfuA includes two cysteine residues that are required for the in vitro assembly of [Fe-S] clusters within the NifU C-terminal domain. Like NifU, NfuA also appears to be a modular protein because the amino acid sequence within its N-terminal region shares some sequence similarity with another protein involved in [Fe-S] protein maturation designated IscA7. The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur [Fe-S] proteins in cyanobacteria and chloroplasts8.7. Bandyopadhyay, S.; Naik, S.G.; O’Carroll, I.P.; Huynh, B.; Dean, D. R.; Johnson, M.K.; Dos Santos, P.C., J. Biol. Chem., 2008, 283, 14092.
8. Jin, Z.; Heinnickel, M.; Krebs, C.; Shen, G.; Golbeck, J.H.; Bryant, D.A., J. Biol. Chem., 2008, 283, 28426.
Education
August 2006
May 2000
April 1994 |
Ph.D. in Chemistry: Bioinorganic Chemistry, Michigan State University, East Lansing, MI, Research Preceptor: Joan B. Broderick, Ph.D., Dissertation title: Investigating the Interactions of S- adenosylmethionine andPyruvate Formate-Lyase-Activating Enzyme: A Radical Activation M.S. in Chemistry: Inorganic Chemistry, University of Florida, Gainesville, FL, Research Preceptor: Michael J. Scott, Ph.D., Thesis title : Synthesis of a Tris Aryloxide-Based Carbamoylmethylphosphine Oxide (CMPO) Ligand for Heavy Metal Ions Extraction B.S. Chemistry: Bioinorganic Chemistry, University of the Philippines, Quezon City, Philippines, Research Preceptor: Titos Anacleto O. Quibuyen, Ph.D., Thesis title: Glucopyranosyl Caproate: Synthesis and Bioassay |
|
Publications
Ye, H.; Jeong, S.Y.; Ghosh, M.C.; Kovtunovych, G.; Silvestri, L.; Ortillo, D.; Uchida, N.; Tisdale, J.; Camaschella, C.; Rouault, T.A., Glutaredoxin 5 deficiency causes sideroblastic anemia by specifically impairing heme biosynthesis and depletingcytosolic iron in human erythroblasts, J. Clin. Inv., 2010, 120, 1749-1761.
Yang, J.; Naik, S.; Ortillo, D.; Garcia, R.; Li, M.; Broderick, W.E.; Huynh, B.H.; Broderick, J.B., The Iron- Sulfur Cluster of Pyruvate Formate-Lyase Activating enzyme in Whole Cells: Cluster Interconversions and a Valence-localized [4Fe-4S]2+ State, Biochemistry, 2009, 49, 9234-9241.
Mulder, D.W.; Ortillo, D.O.; Gardenghi, D.J.; Naumov, A.V.; Ruebush, S.S.; Szilagyi, R.K.; Huynh, B.; Broderick, J.B.; Peters, J.W., Activation of HydAΔEFG Requires a Preformed [4Fe-4S] Cluster, Biochemistry, 2009, 48, 6240-6248.
Murray, L.J.; Naik, S.; Ortillo, D. O.; Garcia-Serres, R.; Lee, J.K.; Huynh, B.H.; Lippard, S.J.,
Characterization of the Arene-Oxidizing Intermediate in TOMOH as a Diiron(III) Species, J. Am. Chem. Soc., 2007, 129, 14500- 14510.
Walsby, C.J.; Ortillo, D.O.; Yang, J.; Nnyepi, M.R.; Broderick, W.E.; Hoffman, B.N.; Broderick, J.B.,
Spectroscopic Approaches to Elucidating Novel Iron-Sulfur Chemistry in the “Radical-SAM” Protein Superfamily, Inorganic Chemistry, 2005, 44, 727-741.
Broderick, J.B.; Walsby, C.; Broderick, W.E.; Krebs, C.; Hong, W.; Ortillo, D.; Cheek, J.; Huynh, B.H.;
Hoffman, B.M., Paramagnetic Resonance in Mechanistic Studies of Fe-S/radical Enzymes: Paramagnetic Resonance of Metallobiomolecules; Telser, J. Ed., American Chemical Society, Washington, DC, 2003, pp 113-127.
Walsby, C.J.; Ortillo, D.O.; Broderick, W.E.; Broderick, J.B.; Hoffman, B.M., An Anchoring Role for Fe-S clusters: Chelation of the Amino Acid Moiety of S-adenosylmethionine to the Unique Iron Site of the [4Fe-4S] Cluster of Pyruvate Formate-Lyase Activating Enzyme, J. Am. Chem. Soc., 2002, 124, 11270-11271.
Walsby, C.J.; Hong, W.; Broderick, W.E.; Cheek, J.; Ortillo, D.; Broderick, J.B.; Hoffman, B.M., Electron-Nuclear Double Resonance Spectroscopic Evidence that S-adenosyl-methionine Binds in Contact with the Catalytically Active [4Fe-4S]+ Cluster of Pyruvate Formate-Lyase Activating Enzyme, J. Am. Chem. Soc., 2002, 124, 3143-3151.
