Dr. James R. Kincaid

Dr. James R. Kincaid
Dr. James R. KincaidMarquette University

Wehr Chemistry Building, 501

MilwaukeeWI53201United States of America
(414) 288-3539


Raman and Time-Resolved Raman Spectroscopy


Ph.D., Marquette University

B.S., Xavier University

Research Interests

The general area of interest to our group is best stated as the structural characterization of reactive heme enzyme intermediates. In recent years the main focus has been on the crucial intermediates encountered in the enzymatic cycles ofCytochromes P450, with special emphasis on steroidogenic P450s responsible for steroid hormone biosynthesis, including CYP11A1, CYP17 and CYP19. One of the most powerful spectroscopic techniques for undertaking such studies is resonance Raman (rR) spectroscopy and our lab is especially well equipped to perform these experiments. Given that the key intermediates are so reactive, special experimental approaches are needed to generate and trap these fleeting species. Referring to Scheme 1, the essential problem is to induce electron transfer to the trapped dioxygen adduct, while controlling the facile associated proton transfer reactions.

Scheme 1

Scheme 1

One effective approach is cryoradiolysis is illustrated in Scheme 2,wherein the frozen trapped dioxygen intermediate is irradiated with ϒ rays from a 60Co source at Notre dame Radiation Lab or at the University of Illinois, the home laboratory of our long-time collaborator, Professor Stephen Sligar.


Scheme 2

Such irradiated samples are transported back to our labs and interrogated by rR; employing both the natural abundance (16O2) and corresponding 18O2 isotopomer, one can generate difference spectra, as shown in the Figure below, which permit identification of the ν(O-O) and ν(Fe-O) vibrational modes of the key Fe-O-O molecular fragment of the dioxygen and ferric peroxo- intermediates. Then, upon careful annealing, controlled proton transfer is facilitated so as to form the next, ferric hydroperoxo-, intermediate. During the past few years this powerful combination of methods has been applied to study crucial intermediates in the enzymatic cycles of Cytochromes P450 and mammalian peroxidases.


Scheme 3

Research Group Members

Dr. Yilin Liu, Postdoctoral Fellow

Dr. Remigio Usai, Postdoctoral Fellow

Mr. Yuanqi Jing, Graduate Student


Professor Aditi Das (U. Illinois)

Professor Patrick Farmer (Baylor University)

Dr. Anabella Ivancich (Marseille, France)

Professor Andrew Munro (Manchester, England)

Professor Stephen Sligar (U. Illinois)

Professor Thomas Spiro (U. Washington)

Professor Robert Tuckey (U. Western Australia)

Professor Lucy Waskell (U. Michigan)

Selected Publications

“Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian Cytochrome P450” Mak, P. J.; Denisov, I. G.; Grinkova, Y. V.; Sligar, S. G.; Kincaid, J. R. J. Am. Chem. Soc. 2011, 133, 1357-1366. DOI:10.1021/ja105869p

 “Effective methods for preparation and characterization of HNO-adducts of heme proteins” Czarnecki, K.; Kincaid, J. R. J. Raman Spectrosc. 2012, 43, 1343-1345. DOI:10.1002/jrs.4046

“Experimental Documentation of the Structural Consequences of Hydrogen-Bonding Interactions to the Proximal Cysteine of a Cytochrome P450” Mak, P. M.; Yang, Y.; Im, S-C; Waskell, L. W.; Kincaid, J. R. Angew. Chem. Int. Ed. 2012, 51, 10403-10407. DOI:10.1002/anie.201205912

 “Differential Hydrogen Bonding in Human CYP17 Dictates Hydroxylation versus Lyase Chemistry” Gregory, M.; Mak, P. J.; Sligar, S.G.; Kincaid, J. R. Angew. Chem. Int. Ed. 2013, 52, 5342-5345. DOI:10.1002/anie.201300760

 “Using resonance Raman cross-section data to estimate the spin-state populations of cytochromes P450” Mak, P. J.; Zhu, Q.; Kincaid, J. R. J. Raman Spectrosc. 2013, 44, 1792-1794. DOI 10.1002/jrs.4401.

 “Resonance Raman determination of vinyl group orientations in different forms of myoglobins” Rwere, F.; Mak, P. J.; Kincaid, J. R. J. Raman Spectrosc., 2014 45, 97-104. DOI 10.1002/jrs.4419

 “Resonance Raman spectroscopy reveals that substrate structure selectively impacts the heme-bound diatomic ligands of CYP17” Mak, P. J.; Gregory, M.; Sligar, S. G.; Kincaid, J. R.  Biochemistry, 2014 53, 90-100. DOI: 10.1021/bi4014424

 “Resonance Raman spectroscopy of the oxygenated intermediates of CYP19 implicates a Compound I intermediate in the final lyase step” Mak, P. J.; Luthra, A.; Sligar, S. G.; Kincaid; J. R., J. Am. Chem. Soc. 2014 136, 4825-4828. DOI 10.1021/ ja500054c

“Differential Control of Heme Reactivity in Alpha and Beta Subunits of Hemoglobin: A Combined Raman Spectroscopic and Computational Study” Jones, E.; Monza, E.; Balakrishnan, G.; Blouin, G.; Mak, P. J.; Zhu, Q.; Kincaid, J. R.; Guallar, V.; Spiro, T. G. J. Am. Chem. Soc. 2014 136, 10325-10339. DOI 10.1021/ja503328a

“Resonance Raman spectroscopy reveals pH-dependent active site structural changes of Lactoperoxidase Compound 0 and its ferryl heme O-O bond cleavage products” Mak, P. M.; Thammawichai, W.; Wiedenhoeft; D.; Kincaid, J. R. J. Am. Chem. Soc. 2015 137, 349-361. DOI 10.1021/ja5107833

“Unveiling the crucial intermediates in androgen production" Mak, P. J.; Gregory, M. C.; Denisov, I. G.; Sligar, S. G.; Kincaid, J. R. Proc. Nat’l. Acad. Sci. (U.S.A.) 2015 112, 15856-15861. DOI 10.10731/pnas.1519376113

“The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4” Denisov, I. G., Mak, P. J.; Grinkova, Y. V.; Bastien, D.; Berube, G.; Sligar, S. G.; Kincaid, J. R. J. Inorg. Biochem. DOI 10.1016/j.jinorgbio. 2015.12.019; in press.

Faculty & Staff


Department of Chemistry

Todd Wehr Chemistry, 101
1414 W Clybourn St.
Milwaukee, WI 53233
(414) 288-3515

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