The general long term goals of the research program are to gain a molecular level insight into the impressive range of functional diversity displayed by various heme proteins. These substances are of critical importance in human physiology and general biology processes. In facilitating many crucial biochemical processes, including oxygen storage and transport, electron transfer, elimination of reactive peroxides, synthesis of hormones and elimination of accumulated environmental toxins, the inherently diverse chemical reactivity of the heme sites are effectively regulated by intramolecular interactions with the associated polypeptides and controlled further by intermolecular interactions with other proteins or small regulatory molecules. In order to fully understand the pertinent relationship of structure to function, it is important to acquire structural information for not only the stable terminal states in a reaction, but also for the fleeting reaction intermediates. This research program uses powerful resonance Raman and time-resolved resonance Raman techniques applied to native and strategically manipulated heme proteins to reveal the molecular basis for modulation of heme group reactivity. The specific systems to be studied in the research being proposed here are two important sub-classes of cytochrome P450 enzymes involved in human physiology. One class, the steroidogenic P450s catalyze the chemical conversions of critical endogenous substances to form essential steroid hormones. The other subclass metabolizes exogenous substances including pharmaceuticals and environmental pollutants, preparing them for elimination.

Public Health Relevance

Heme proteins are molecules that are of prime importance for proper functioning of many biochemical processes important to human physiology and other forms of life. The main task facing heme protein researchers is to be able to gain insight into the structure of the protein active site, where the key chemical reactions occur, such insight being expected to be very useful in designing treatments for some diseases. The resonance Raman technique being used in this work is one of the most powerful methods to acquire this critical information.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Wehrle, Janna P
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Marquette University
Schools of Arts and Sciences
United States
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Zhu, Qianhong; Mak, Piotr J; Tuckey, Robert C et al. (2017) Active Site Structures of CYP11A1 in the Presence of Its Physiological Substrates and Alterations upon Binding of Adrenodoxin. Biochemistry 56:5786-5797
Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V et al. (2016) The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4. J Inorg Biochem 158:77-85
Mak, Piotr J; Gregory, Michael C; Denisov, Ilia G et al. (2015) Unveiling the crucial intermediates in androgen production. Proc Natl Acad Sci U S A 112:15856-61
Mak, Piotr J; Luthra, Abhinav; Sligar, Stephen G et al. (2014) Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound i intermediate in the final lyase step. J Am Chem Soc 136:4825-8
Mak, Piotr J; Gregory, Michael C; Sligar, Stephen G et al. (2014) Resonance Raman spectroscopy reveals that substrate structure selectively impacts the heme-bound diatomic ligands of CYP17. Biochemistry 53:90-100
Mak, Piotr J; Zhu, Qianhong; Kincaid, James R (2013) Using resonance Raman cross-section data to estimate the spin state populations of Cytochromes P450. J Raman Spectrosc 44:1792-1794
Gregory, Michael; Mak, Piotr J; Sligar, Stephen G et al. (2013) Differential hydrogen bonding in human CYP17 dictates hydroxylation versus lyase chemistry. Angew Chem Int Ed Engl 52:5342-5
Mak, Piotr J; Yang, Yuting; Im, Sangchoul et al. (2012) Experimental documentation of the structural consequences of hydrogen-bonding interactions to the proximal cysteine of a cytochrome P450. Angew Chem Int Ed Engl 51:10403-7
Mak, Piotr J; Denisov, Ilia G; Grinkova, Yelena V et al. (2011) Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian cytochrome P450. J Am Chem Soc 133:1357-66