Research
Structure-function studies of heme proteins - Iron protoporphyrin IX (heme b, protoheme) is a highly versatile prosthetic group used by many proteins to support their diverse range of chemical activity. From “simple” oxygen transport (globins), through peroxidase reactivity (hydroperoxidases, peroxygenases) to the impressive reductive activation of atmospheric oxygen (cytochrome P450s, nitric oxide synthases and cytochrome c oxidase), all these heme proteins utilize basically the same prosthetic group, protoheme, or its structurally similar analogues. The main goal of our research is to understand how these structurally different proteins effectively manipulate the inherent reactivity of the same heme macrocycle, and its bound endogenous and exogenous ligands, in order to magnify a particular inherent function, while suppressing others.
Our studies focus on elucidating structural features that control the enzymatic reactivity of heme proteins, including variations of the heme active site or environmental conditions, including the nature of the interactions with redox partner proteins or cofactors. These goals are achieved using mainly resonance Raman (rR) spectroscopy which permits detailed characterization of even very subtle, but functionally significant, active site structural perturbations. Application of an innovative combination of techniques, such as coupling of rR spectroscopy with the cryoradiolysis method, permit interrogation of key enzymatic intermediates including unstable dioxy, peroxo- and hydroperoxo- species, as well as the crucial intermediates that arise from attack of these oxy-intermediates on substrates.
adopted from: Cytochrome P450: Structure, Mechanism, and Biochemistry, Ortiz de Montellano, P. R. (Ed.), Kluwer Academic/Plenum Publisher, New York, 2005, 149-182.
Techniques
Resonance Raman - Resonance Raman (rR) spectroscopy is a powerful probe of heme proteins, not only for the equilibrated terminal states, but also for the fleeting intermediates. The heme marker modes respond to changes in oxidation- or spin-states of the central iron in well-established and documented ways. The low frequency modes document changes in heme planarity and in protein interactions with heme periphery. This is important, because the presence of the propionic acid and potentially conjugated vinyl peripheral substituents are considered as effective structural determinants of heme reactivity.
Of special significance, rR experiments offer important insight into the key linkages between the heme prosthetic group and the endogenous ligands provided by the associated protein or exogenous ligands; i.e., it is possible to efficiently enhance internal modes of Fe-L or Fe-XY fragments, where L refers to a protein-based endogenous axial ligand (His, Cys) and XY indicates a exogenous ligand, including dioxygen, carbon monoxide or nitric oxide.
Check these papers:
1. “Resonance Raman Spectroscopy as a Structural Probe of the Cytochrome P450 Enzymatic Cycle”, Mak, P. J. in Handbook of Porphyrin Science (Kadish, K. M., Smith, K.M., Guilard, R., Ed.), 2016, pp 1-120, World Scientific, N.J.
2. “Resonance Raman spectra of heme proteins and model compounds”, Kincaid, J. R. in Porphyrin Handbook (Kadish, K. M., Smith, K.M., Guilard, R., Ed.),2000, pp 225-291, Academic Press, N.Y.
3. “Resonance Raman Spectra of Heme Proteins and Model Compounds”, Spiro, T. G., (Ed.) 1988, Wiley and Sons, N.Y.
Cryoradiolysis - Coupling of rR spectroscopy with cryoradiolysis method provides an effective method to generate, trap and structurally characterize otherwise elusive enzymatic intermediates. The cryoradiolysis approach involves irradiation of the freeze-trapped dioxygen adducts with gamma-rays. The free-electrons and organic radicals are produced, the electrons being mobile, while other movements, including proton transport, are more restricted at 77 K. The freeze-trapped Fe(III)-O-O peroxo fragment is generated and can be now spectroscopically characterized, including rR spectroscopy. Careful annealing to higher temperatures permits proton transfer and trapping of the hydroperoxo- species, while further annealing can lead to delivery of another proton to facilitate the O-O bond cleavage, with generation of Compound I or other reactive intermediate.
Interesting read:
1. “Cryoradiolysis and cryospectroscopy for studies of heme-​oxygen intermediates in cytochromes P450”, Denisov, I. G.; Grinkova, Y. V.; Sligar, S. G. Meth. Mol. Biol. 2012, 875, 375-391.
2. “Active intermediates in heme monooxygenase reactions as revealed by cryoreduction​/annealing, EPR​/ENDOR studies”, Davydov, R.; Hoffman, B. M. Arch. Biochem. Biophys. 2011, 507, 36-43.
3. “Electron spin and electron nuclear double resonance of the [FeO2]- [ferrite] center from irradiated oxyhemo- and oxymyoglobin”, Kappl, R.; Hoehn-Berlage, M.; Huettermann, J.; Bartlett, N.; Symons, M. C. R. Biochim. Biophys. Acta 1985, 827, 327-343.