This project aims to extend our understanding of the structure, function, and dynamics of heme proteins such as cytochrome P450, mammalian peroxidases, nitric oxide synthase, soluble guanylate cyclase, nitrophorin, and cytochrome c. These proteins are involved in broad array of catalytic, signaling, and electron transport processes and are capable of an amazingly broad range of functions, even when the heme axial ligands are identical. This indicates that the protein architecture, and its influence on the heme structure, plays an important functional role. By using coherence spectroscopy, a femtosecond optical """"""""pump-probe"""""""" technique, the """"""""soft"""""""" out-of-plane (OOP) low-frequency vibrational modes of the heme can be excited and analyzed even in an aqueous environment. These vibrational modes have not been documented previously because they are difficult to access using traditional spectroscopic methods. They fall in the region of ambient thermal excitations (<200cm-1 ~300K) and are therefore most likely to be utilized as reaction coordinates by proteins. The observed coherence spectral intensities of these """"""""soft"""""""" modes depend upon the magnitude of the heme structural distortions that are induced by the protein architecture. These OOP heme motions are functionally significant, as demonstrated by the importance of the heme """"""""doming"""""""" mode in the diatomic ligand binding reaction. The rich spectrum of the low-frequency heme motions is just beginning to be appreciated, as a wider variety of proteins and model compounds is examined. This project aims to explore the functional role of both static distortions and thermally excited low-frequency vibrations in heme proteins. Distortions (such as heme """"""""ruffling"""""""" and """"""""saddling"""""""") that alter the electronic orbital interactions between the iron atom and its surrounding molecular framework are hypothesized to affect the redox potential of the metal center. Vibrational motions along these same, thermally accessible, OOP coordinates are excellent candidates to mediate and control electron transfer. Coherence spectroscopy is uniquely positioned to probe these modes in aqueous solution. For example, we will examine electron transfer partners, such as Pdx and CYP101, in order to monitor changes in the low frequency spectrum that occur when the protein complex is formed. The low frequency modes of Fe-S proteins will also be examined. Kinetic probes on ultrafast timescales, stretching over 10 decades in time, will be used to study the rapid time-scale, non-equilibrium processes, that take place immediately following the electronic rearrangements associated with biochemical reactions. For example, the two geminate phases for oxygen rebinding to the heme in myoglobin exhibit very different Arrhenius prefactors, suggesting that the entropic barrier for recombination is time dependent. Such non-equilibrium processes will be studied to learn if they allow heme proteins to enhance discrimination between different classes of diatomic ligands.

Public Health Relevance

This project has a wide range of health related implications involving heme and iron-sulfur metalloproteins. Many metabolic disease states involve disrupted catalytic, signaling, and/or electron transport processes that involve such proteins. All biological processes that involve molecular electron transport, and/or utilize the heme or iron-sulfur cofactors, are related to this research. A fundamental understanding of how the protein architecture interacts with the metal co-factors, and how this translates into vibrational dynamics and function at the molecular level, will lead to deeper insights for those concerned with the treatment of metabolic disorders at any level. Basic research on metalloproteins is essential to our composite understanding of the human body. This project is deeply involved with investigations of these systems at a fundamental level.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Sechi, Salvatore
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Northeastern University
Schools of Arts and Sciences
United States
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Karunakaran, Venugopal; Sun, Yuhan; Benabbas, Abdelkrim et al. (2014) Investigations of the low frequency modes of ferric cytochrome c using vibrational coherence spectroscopy. J Phys Chem B 118:6062-70
Sun, Yuhan; Zeng, Weiqiao; Benabbas, Abdelkrim et al. (2013) Investigations of heme ligation and ligand switching in cytochromes p450 and p420. Biochemistry 52:5941-51
Zeng, Weiqiao; Sun, Yuhan; Benabbas, Abdelkrim et al. (2013) Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase. J Phys Chem B 117:4042-9
Sun, Yuhan; Karunakaran, Venugopal; Champion, Paul M (2013) Investigations of the low-frequency spectral density of cytochrome c upon equilibrium unfolding. J Phys Chem B 117:9615-25
Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan et al. (2012) Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA. J Biol Chem 287:21729-40
Barabanschikov, Alexander; Demidov, Alexander; Kubo, Minoru et al. (2011) Spectroscopic identification of reactive porphyrin motions. J Chem Phys 135:015101
Karunakaran, Venugopal; Benabbas, Abdelkrim; Youn, Hwan et al. (2011) Vibrational coherence spectroscopy of the heme domain in the CO-sensing transcriptional activator CooA. J Am Chem Soc 133:18816-27
Karunakaran, Venugopal; Denisov, Ilia; Sligar, Stephen G et al. (2011) Investigation of the low frequency dynamics of heme proteins: native and mutant cytochrome P450(cam) and redox partner complexes. J Phys Chem B 115:5665-77
Karunakaran, Venugopal; Benabbas, Abdelkrim; Sun, Yuhan et al. (2010) Investigations of low-frequency vibrational dynamics and ligand binding kinetics of cystathionine beta-synthase. J Phys Chem B 114:3294-306
Benabbas, Abdelkrim; Ye, Xiong; Kubo, Minoru et al. (2010) Ultrafast dynamics of diatomic ligand binding to nitrophorin 4. J Am Chem Soc 132:2811-20

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