Electron Transfer in Iron and Copper Proteins

Summary

Principal Investigator: Jay R Winkler
Abstract: A great many biological energy transduction pathways depend upon the rapid movement of electrons or holes over long distances (>30 [unreadable]) through proteins. Many redox enzymes, particularly those involved in oxygen activation and production, require the transfer of holes at high potentials where the sidechains of redox-active amino acids can become involved. Stringent design requirements must be met to transport charges rapidly and efficiently along specific pathways and prevent the off- path diffusion of redox equivalents and the disruption of energy flow. In this research program, the molecular parameters that control multistep electron tunneling through tryptophan (Aim 1) and tyrosine (Aim 2) radicals will be examined. Several azurin mutants in which tryptophan residues lie between the Cu active site and a surface-attached photosensitizer will be prepared. Kinetics measurements will be used to elucidate the roles of redox-site separation distance, driving force, and tryptophan environment on the dynamics of long-range charge transport. Tyrosine is postulated to mediate long-range hole transport in several redox enzymes. Azurin mutants in which tyrosine residues are placed to serve as mediators of long-range electron transfer will be prepared and studied. Mutant proteins in which proton accepting residues are within hydrogen-bonding distance of the intermediate tyrosine residue also will be prepared. These proximal proton acceptors are expected to facilitate long-range hopping via tyrosine radicals. Amino-acid sidechain radicals often are found in enzymes involved in oxygen activation and formation chemistry. Important members of this class of enzymes are the heme-thiolate mono- oxygenases such as cytochrome P450 and nitric oxide synthases (NOS). These enzymes utilize molecular oxygen to effect a wide range of hydroxylation reactions involving organic substrates. Despite extensive investigations in many laboratories, questions remain regarding the nature of the active hydroxylating reagent in these enzymes: ferryl species (i.e., Fe-oxo Compounds I and II) are often invoked. Protein labeling and laser flash-quench photochemistry will be used to develop new insights into the reactive intermediates in heme-thiolate mono-oxygenases. Rather than treating the enzymes with reactive oxygen compounds, photochemically generated one-electron, outer-sphere oxidants covalently bound to the protein periphery will be used to remove electrons from the resting enzyme and produce ferryl compounds. Efforts will focus on oxidative generation of Compounds I and II in cytochromes P450 (Aim 3) and a bacterial enzyme that exhibits NOS-like reactivity (Aim 4).
Funding Period: 1979-05-01 - 2015-04-30
more information: NIH RePORT

Top Publications

  1. pmc Long-range electron tunneling
    Jay R Winkler
    Beckman Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, United States
    J Am Chem Soc 136:2930-9. 2014
  2. pmc Snapshots of a protein folding intermediate
    Seiji Yamada
    Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA
    Proc Natl Acad Sci U S A 110:1606-10. 2013
  3. pmc Catalysis of dioxygen reduction by Thermus thermophilus strain HB27 laccase on ketjen black electrodes
    Peter Agbo
    California Institute of Technology, Pasadena, California, USA
    J Phys Chem B 117:527-34. 2013
  4. pmc Photoexcitation of tumor-targeted corroles induces singlet oxygen-mediated augmentation of cytotoxicity
    Jae Youn Hwang
    Department of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA, USA
    J Control Release 163:368-73. 2012
  5. pmc Inner- and outer-sphere metal coordination in blue copper proteins
    Jeffrey J Warren
    Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA
    J Inorg Biochem 115:119-26. 2012
  6. pmc Spin delocalization over type zero copper
    Alexey Potapov
    Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel
    Inorg Chem 51:4066-75. 2012
  7. pmc Investigating photoexcitation-induced mitochondrial damage by chemotherapeutic corroles using multimode optical imaging
    Jae Youn Hwang
    Minimally Invasive Surgical Technologies Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard D6061, Los Angeles, California 90048, USA
    J Biomed Opt 17:015003. 2012
  8. pmc Gating NO release from nitric oxide synthase
    Charlotte A Whited
    Beckman Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
    J Am Chem Soc 134:27-30. 2012
  9. pmc A mechanistic study of tumor-targeted corrole toxicity
    Jae Youn Hwang
    Department of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, California, United States
    Mol Pharm 8:2233-43. 2011
  10. pmc Ratiometric spectral imaging for fast tumor detection and chemotherapy monitoring in vivo
    Jae Youn Hwang
    University of Southern California, Department of Biomedical Engineering, Los Angeles, California 90089, USA
    J Biomed Opt 16:066007. 2011

Research Grants

  1. Fast Kinetic Investigations of Nitric Oxide Synthase
    Raymond M Esquerra; Fiscal Year: 2013
  2. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013
  3. Ru(II)-diimine labeled P450 mutants for the selective hydroxylation of substrate
    Lionel E Cheruzel; Fiscal Year: 2013
  4. Synthetic Modeling of Copper Protein Active Sites
    William B Tolman; Fiscal Year: 2013
  5. Reactivity of Manganese and Iron Metalloenzyme Models
    David P Goldberg; Fiscal Year: 2013
  6. BIOINORGANIC COPPER COORDINATION CHEMISTRY
    Kenneth D Karlin; Fiscal Year: 2013
  7. Structural bases of the functions of RNA-protein machines
    THOMAS ARTHUR STEITZ; Fiscal Year: 2013
  8. IPF Fibroblast Phenotype
    Craig A Henke; Fiscal Year: 2013
  9. Protein Dynamics in Enzymatic Catalysis
    Robert Callender; Fiscal Year: 2013

Detail Information

Publications49

  1. pmc Long-range electron tunneling
    Jay R Winkler
    Beckman Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, United States
    J Am Chem Soc 136:2930-9. 2014
    ..The range of phenomena that depends on long-range electron tunneling continues to expand, providing new challenges for both theory and experiment. ..
  2. pmc Snapshots of a protein folding intermediate
    Seiji Yamada
    Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA
    Proc Natl Acad Sci U S A 110:1606-10. 2013
    ..The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein...
  3. pmc Catalysis of dioxygen reduction by Thermus thermophilus strain HB27 laccase on ketjen black electrodes
    Peter Agbo
    California Institute of Technology, Pasadena, California, USA
    J Phys Chem B 117:527-34. 2013
    ....
  4. pmc Photoexcitation of tumor-targeted corroles induces singlet oxygen-mediated augmentation of cytotoxicity
    Jae Youn Hwang
    Department of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, CA, USA
    J Control Release 163:368-73. 2012
    ....
  5. pmc Inner- and outer-sphere metal coordination in blue copper proteins
    Jeffrey J Warren
    Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA
    J Inorg Biochem 115:119-26. 2012
    ..In this tribute to Hans Freeman, we review investigations that have advanced the understanding of how inner-sphere and outer-sphere coordination affects biological Cu properties...
  6. pmc Spin delocalization over type zero copper
    Alexey Potapov
    Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel
    Inorg Chem 51:4066-75. 2012
    ..The increased spin delocalization over the D112 carboxylate in type zero mutants compared to type 2 C112D suggests that electron transfer paths involving this residue are enhanced...
  7. pmc Investigating photoexcitation-induced mitochondrial damage by chemotherapeutic corroles using multimode optical imaging
    Jae Youn Hwang
    Minimally Invasive Surgical Technologies Institute, Cedars Sinai Medical Center, 8700 Beverly Boulevard D6061, Los Angeles, California 90048, USA
    J Biomed Opt 17:015003. 2012
    ..Our demonstration of HerGa phototoxicity opens the way for development of new methods of cancer intervention using tumor-targeted corroles...
  8. pmc Gating NO release from nitric oxide synthase
    Charlotte A Whited
    Beckman Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
    J Am Chem Soc 134:27-30. 2012
    ....
  9. pmc A mechanistic study of tumor-targeted corrole toxicity
    Jae Youn Hwang
    Department of Biomedical Sciences, Cedars Sinai Medical Center, Los Angeles, California, United States
    Mol Pharm 8:2233-43. 2011
    ..Altogether, these studies identify a mechanistic pathway in which early endosomal escape enables HerGa-induced superoxide generation leading to cytoskeletal and mitochondrial damage, thus triggering downstream cell death...
  10. pmc Ratiometric spectral imaging for fast tumor detection and chemotherapy monitoring in vivo
    Jae Youn Hwang
    University of Southern California, Department of Biomedical Engineering, Los Angeles, California 90089, USA
    J Biomed Opt 16:066007. 2011
    ....
  11. pmc Light-triggered modulation of cellular electrical activity by ruthenium diimine nanoswitches
    Joyce G Rohan
    Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089 2821, USA
    ACS Chem Neurosci 4:585-93. 2013
    ..While the present work is focused on ruthenium diimine complexes, the findings point more generally to broader application of other transition metal complexes to mediate light-induced biological changes...
  12. pmc A Euclidean perspective on the unfolding of azurin: chain motion
    Harry B Gray
    Beckman Institute, California Institute of Technology, Pasadena, CA, 91125, USA
    J Biol Inorg Chem 19:555-63. 2014
    ..This static behavior is a consequence of a dynamic balance between the competing motion of two segments, residues 72-81 and 84-91. ..
  13. pmc Enhancement of anion binding in lanthanide optical sensors
    Morgan L Cable
    Jet Propulsion Laboratory, Pasadena, California 91109, United States
    Acc Chem Res 46:2576-84. 2013
    ..Ancillary ligands also can reduce the impact of interfering species such as phosphate commonly found in environmental and physiological samples. ..
  14. pmc Tryptophan-accelerated electron flow across a protein-protein interface
    Kana Takematsu
    Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States
    J Am Chem Soc 135:15515-25. 2013
    ..These findings on interfacial electron hopping in (Re126W122Cu(I))2 shed new light on optimal redox-unit placements required for functional long-range charge separation in protein complexes. ..
  15. pmc Intrachain contact dynamics in unfolded cytochrome cb562
    Nicole D Bouley Ford
    Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States
    J Phys Chem B 117:13206-11. 2013
    ..For other labeling sites within the stable loop, quenching rates were modestly reduced compared to the open chain polymer. ..
  16. pmc Electron flow through nitrotyrosinate in Pseudomonas aeruginosa azurin
    Jeffrey J Warren
    Beckman Institute and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA
    J Am Chem Soc 135:11151-8. 2013
    ..Our analysis of transient optical spectroscopic experiments indicates that hopping via NO2YO(-) enhances Cu(I) oxidation rates over single-step ET by factors of 32 (RuH107NO2YO(-)109), 46 (RuH126NO2YO(-)122), and 13 (RuH124NO2YO(-)122). ..
  17. pmc A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo
    Pedro S Coelho
    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA
    Nat Chem Biol 9:485-7. 2013
    ....
  18. pmc Phototriggering electron flow through Re(I)-modified Pseudomonas aeruginosa azurins
    Ana Maria Blanco-Rodriguez
    Queen Mary University of London, School of Biological and Chemical Sciences, Mile End Road, London E1 4NS, UK
    Chemistry 17:5350-61. 2011
    ..Re(I)(CO)(3)(dmp(.-))] potentials that result in very rapid electron interchange and (3)CT-(3)CS energetic proximity, are the main factors responsible for the unique ET behavior of [Re(I)(dmp)(W122)]-containing azurins...
  19. pmc Mass spectrometric characterization of oligomers in Pseudomonas aeruginosa azurin solutions
    Lucie Sokolova
    Institute of Physical and Theoretical Chemistry, Goethe University, Frankfurt am Main, Germany
    J Phys Chem B 115:4790-800. 2011
    ....
  20. pmc Electron transfer reactivity of type zero Pseudomonas aeruginosa azurin
    Kyle M Lancaster
    Institute of Analytical Chemistry, University of Copenhagen, 2100 Copenhagen, Denmark
    J Am Chem Soc 133:4865-73. 2011
    ..9-1.1 eV) slightly above that for type 1 (0.7-0.8 eV), but substantially smaller than that for type 2 (>2 eV), consistent with XAS and EXAFS data that reveal minimal type zero site reorientation during redox cycling...
  21. pmc Electrostatic effects on funneled landscapes and structural diversity in denatured protein ensembles
    Patrick Weinkam
    Center for Theoretical Biological Physics and Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
    Proc Natl Acad Sci U S A 106:1796-801. 2009
    ..Comparing simulations to detailed experimental data on several labeling sites allows us to quantify the dominant forces in denatured protein ensembles...
  22. pmc High-potential C112D/M121X (X = M, E, H, L) Pseudomonas aeruginosa azurins
    Kyle M Lancaster
    Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA
    Inorg Chem 48:1278-80. 2009
    ..The high potential and low axial hyperfine splitting (Cu(II) electron paramagnetic resonance A( parallel)) of the C112D/M121L protein are remarkably similar to features normally associated with type 1 copper centers...
  23. pmc Synchronous vs asynchronous chain motion in alpha-synuclein contact dynamics
    Kristopher G Urie
    Department of Chemistry, DePaul University, 243 South Wabash Ave, Chicago, Illinois 60604 2301, USA
    J Phys Chem B 113:522-30. 2009
    ..The additional deviations from random coil behavior in alpha-syn likely arise from clustering of hydrophobic residues in the disordered polypeptide...
  24. ncbi Tryptophan-accelerated electron flow through proteins
    Crystal Shih
    Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA
    Science 320:1760-2. 2008
    ....
  25. pmc Enantiomer-specific binding of ruthenium(II) molecular wires by the amine oxidase of Arthrobacter globiformis
    David B Langley
    School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia
    J Am Chem Soc 130:8069-78. 2008
    ..Both the Lambda and Delta isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases...
  26. pmc Copper(II) binding to alpha-synuclein, the Parkinson's protein
    Jennifer C Lee
    Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 8013, USA
    J Am Chem Soc 130:6898-9. 2008
    ..Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site...
  27. ncbi Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex
    Erik W Debler
    Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA
    Science 319:1232-5. 2008
    ..Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody...
  28. pmc Alpha-synuclein tertiary contact dynamics
    Jennifer C Lee
    Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, Maryland 20892, USA
    J Phys Chem B 111:2107-12. 2007
    ..The contact rates for these large loops indicate much shorter average donor-acceptor separations than expected for a random polymer...
  29. ncbi Spectroscopy and electrochemistry of cytochrome P450 BM3-surfactant film assemblies
    Andrew K Udit
    Department of Chemistry, Occidental College, Los Angeles, California 90041, USA
    J Am Chem Soc 128:10320-5. 2006
    ....
  30. pmc Folding energy landscape of cytochrome cb562
    Tetsunari Kimura
    Beckman Institute, California Institute of Technology, Pasadena, CA 91125 7400, USA
    Proc Natl Acad Sci U S A 106:7834-9. 2009
    ..e., the heme prosthetic group) without the formation of deep misfolded traps...
  31. pmc Nanosecond photoreduction of inducible nitric oxide synthase by a Ru-diimine electron tunneling wire bound distant from the active site
    Charlotte A Whited
    Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA
    J Inorg Biochem 103:906-11. 2009
    ..Photoreduction of an imidazole-bound active-site heme iron in the enzyme-wire conjugate (k(ET) = 2(1) x 10(7) s(-1)) is fully seven orders of magnitude faster than the in vivo process...
  32. ncbi Spectroscopic and redox properties of amine-functionalized K2[OsII(bpy)(CN)4] complexes
    Michael J Ahrens
    Ohmx Corporation, 1801 Maple Avenue, Suite 6143, Evanston, Illinois 60201, USA
    Dalton Trans 40:1732-6. 2011
    ..The pendant amine functional groups and coordinated cyanide ligands are basic sites that can be sequentially protonated, thereby allowing systematic tuning of electrochemical and optical spectroscopic properties...
  33. pmc Photooxidation of cytochrome P450-BM3
    Maraia E Ener
    Beckman Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA
    Proc Natl Acad Sci U S A 107:18783-6. 2010
    ..Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting...
  34. pmc Outer-sphere effects on reduction potentials of copper sites in proteins: the curious case of high potential type 2 C112D/M121E Pseudomonas aeruginosa azurin
    Kyle M Lancaster
    Beckman Institute, California Institute of Technology, Pasadena, California 91125, USA
    J Am Chem Soc 132:14590-5. 2010
    ..These results highlight the role played by outer-sphere interactions, and the structural constraints they impose, in determining the redox behavior of transition metal protein cofactors...
  35. pmc Type-zero copper proteins
    Kyle M Lancaster
    California Institute of Technology, Pasadena, California 91125, USA
    Nat Chem 1:711-5. 2009
    ..Cyclic voltammetric experiments demonstrate that the electron transfer reactivities of type-zero azurins are enhanced relative to that of the corresponding type 2 (C112D) protein...
  36. ncbi Relaxation dynamics of Pseudomonas aeruginosa Re(I)(CO)3(alpha-diimine)(HisX)+ (X = 83, 107, 109, 124, 126)Cu(II) azurins
    Ana Maria Blanco-Rodriguez
    School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom
    J Am Chem Soc 131:11788-800. 2009
    ..Our work demonstrates that time-resolved IR spectroscopy and emission anisotropy of Re(I) carbonyl-diimine complexes are powerful probes of molecular dynamics at or around the surfaces of proteins and protein-protein interfacial regions...
  37. pmc Detection of bacterial spores with lanthanide-macrocycle binary complexes
    Morgan L Cable
    Planetary Science Section, Jet Propulsion Laboratory, 4800 Oak Grove Drive, Pasadena, California 91109, USA
    J Am Chem Soc 131:9562-70. 2009
    ..Out of the eight cases investigated, the Tb(DO2A)(+) binary complex is best for the detection of bacterial spores...
  38. ncbi Excited-state dynamics of structurally characterized [ReI(CO)3(phen)(HisX)]+ (X = 83, 109) Pseudomonas aeruginosa azurins in aqueous solution
    Ana Maria Blanco-Rodriguez
    School of Biological and Chemical Sciences, Queen Mary, University of London, UK
    J Am Chem Soc 128:4365-70. 2006
    ..5 ps); the 250-ps relaxation is attributable to reorientation of D(2)O molecules as well as structural reorganization of the rhenium chromophore and nearby polar amino acids in each of the modified proteins...

Research Grants30

  1. Fast Kinetic Investigations of Nitric Oxide Synthase
    Raymond M Esquerra; Fiscal Year: 2013
    ..Understanding how NOS is regulated and clarifying its catalytic mechanism are crucial both for designing therapies that control NO synthesis and for understanding how compromised NO physiology leads to deleterious health effects. ..
  2. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013
    ..The ultimate goal of all the proposed studies is to define the molecular events that feature in the formation of human prions in order to develop therapeutics that cure the human prion diseases. ..
  3. Ru(II)-diimine labeled P450 mutants for the selective hydroxylation of substrate
    Lionel E Cheruzel; Fiscal Year: 2013
    ..This system will be used as an innovative model of human P450 enzymes, which are responsible for the bioactivation and biodegradation of 75% of all drugs. ..
  4. Synthetic Modeling of Copper Protein Active Sites
    William B Tolman; Fiscal Year: 2013
    ..In addition to aspiring to a deep understanding of copper enzyme structure/function relationships, the proposed work is aimed at developing novel copper chemistry of fundamental significance. ..
  5. Reactivity of Manganese and Iron Metalloenzyme Models
    David P Goldberg; Fiscal Year: 2013
    ..Fundamental information regarding the mechanisms of biomimetic HAT, OAT, ET and related reactions will be obtained. ..
  6. BIOINORGANIC COPPER COORDINATION CHEMISTRY
    Kenneth D Karlin; Fiscal Year: 2013
    ..Potential long-term applications of this basic research include development of enzyme inhibitors and relevant disease therapeutic strategies. ..
  7. Structural bases of the functions of RNA-protein machines
    THOMAS ARTHUR STEITZ; Fiscal Year: 2013
    ..Also of interest will be the ways in which the structures and properties of RNA molecules can be utilized to carry out various biological functions often analogous to those performed by proteins. ..
  8. IPF Fibroblast Phenotype
    Craig A Henke; Fiscal Year: 2013
    ..A major objective of this Program Project is to inform decisions of the IPF Clinical Network by providing information that can be translated into novel therapeutic strategies for IPF. ..
  9. Protein Dynamics in Enzymatic Catalysis
    Robert Callender; Fiscal Year: 2013
    ..The Equipment Core (Core A) supports the specialized comprehensive suite of instrumentation for the Program. The Administrative Core (Core B) administers the Program Project. ..