The energetic foundation of cellular activity and its regulation relies on a string of oxidoreductase proteins in biological oxidative and reductive metabolism and respiratory membrane energy conversion. The mechanisms of many catalytic sites of substrate oxidation-reduction and energy conversion, particularly in mitochondria! respiration, have proven to be difficult to access experimentally due to natural complexity and fragility. They remain poorly understood. Our proposal aims to reveal the natural engineering of the redox- coupled proton exchange and transfer operating at these sites of multi-electron cofactor and substrate oxidation-reduction. Our approach builds on our engineering guidelines for protein electron tunneling, strengthened in the last grant period, to inform the de novo design and assembly of simple and robust alpha- helical proteins intended to serve as protein-based models, maquettes, for multi-electron catalysis. Maquettes will be designed to provide the simplest water-soluble or trans-membrane structures that can capture the functional properties of natural redox centers. Their simplicity and adaptability allow us to investigate catalytic functional problems that remain unsolved in the respiratory chain. Maquettes will be activated with light and electrometric methods in solution and on electrodes to dissect step-by-step the thermodynamics and kinetics of electron transfer and proton exchange. Maquettes will incorporate all key two-electron, multi-proton cofactors/substrates quinone, nicotinamide and flavin, and the two-and four- electron substrate O2. We are positioned to focus on the problem of reversible energy conversion catalysis of hydroquinone-quinone in the Qo site of the cytochrome bd aiming to determine the mechanistic root of medically harmful short-circuits and radical generation. We will extend our maquette creation of a stable O2 ferrous heme state, to examine two- and four-electron O2 reduction and the physiologically important two- electron chemistries of NO and H2O2. With stable and adaptable maquettes, we can exploit physiological chemistry in the development of nanoscale devices. The breakdown of food by oxygen respiration in humans produces all the energy needed for a healthy life and is a central part of cellular regulation. It is normal that there is a steady slow release of oxygen radicals that over time cause cellular damage, aging and disease. Under stress, and in during many surgical procedures bursts of radicals can accelerate these deleterious processes. The research of this grant describes a new way to understand the processes underlying healthy oxidative energy supply and control as well as deleterious radical generation. With progress we will be better predict and track the onset of disease and act to slow it or reverse it.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041048-21
Application #
7738902
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1989-02-01
Project End
2010-11-30
Budget Start
2009-12-01
Budget End
2010-11-30
Support Year
21
Fiscal Year
2010
Total Cost
$355,604
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Lichtenstein, Bruce R; Bialas, Chris; Cerda, José F et al. (2015) Designing Light-Activated Charge-Separating Proteins with a Naphthoquinone Amino Acid. Angew Chem Int Ed Engl 54:13626-9
Anderson, J L Ross; Armstrong, Craig T; Kodali, Goutham et al. (2014) Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette. Chem Sci 5:507-514
Solomon, Lee A; Kodali, Goutham; Moser, Christopher C et al. (2014) Engineering the assembly of heme cofactors in man-made proteins. J Am Chem Soc 136:3192-9
Farid, Tammer A; Kodali, Goutham; Solomon, Lee A et al. (2013) Elementary tetrahelical protein design for diverse oxidoreductase functions. Nat Chem Biol 9:826-833
Raju, Gheevarghese; Capo, Joseph; Lichtenstein, Bruce R et al. (2012) Manipulating Reduction Potentials in an Artificial Safranin Cofactor. Tetrahedron Lett 53:1201-1203
Lichtenstein, Bruce R; Farid, Tammer A; Kodali, Goutham et al. (2012) Engineering oxidoreductases: maquette proteins designed from scratch. Biochem Soc Trans 40:561-6
Lichtenstein, Bruce R; Moorman, Veronica R; Cerda, José F et al. (2012) Electrochemical and structural coupling of the naphthoquinone amino acid. Chem Commun (Camb) 48:1997-9
Dutton, P Leslie; Moser, Christopher C (2011) Engineering enzymes. Faraday Discuss 148:443-8
Zhang, Lei; Anderson, J L Ross; Ahmed, Ismail et al. (2011) Manipulating cofactor binding thermodynamics in an artificial oxygen transport protein. Biochemistry 50:10254-61
Cui, Dongtao; Koder, Ronald L; Dutton, P Leslie et al. (2011) 15N solid-state NMR as a probe of flavin H-bonding. J Phys Chem B 115:7788-98

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