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A solution nuclear magnetic resonance (NMR) spectroscopic study of cyanide-inhibited, substrate-bound complex of uniformly (super 15)N-labeled human heme oxygenase (hHO) was performed for characterizing the active site with respect to the nature and identity of strong hydrogen bonds and the occupation of ordered water molecules within the hydrogen bonding network and an aromatic cluster on the distal side.

The roles of proximal thiolate iron ligand in cytochrome P450 were studied use resonance Raman, UV, circular dichroism and activity measurements.

Noncarane and spiro[2.5]octane are used to determine the mechanisms of hydrocarbon hydroxylation by two mammalian and two bacterial cytochrome P450 enzymes. Results present a consistent pattern of rearrangement that supports a radical rebound mechanism for this set of cytochrome P450 enzymes.

The (super 1)H NMR identifies the water molecules localized within a protein through NOEs between the bulk water signal and individual protein resonances. Water molecules roles can range from simple impediments to rapid ligation, as in globins, to participation in important hydrogen-bonding networks in the catalytic cycles of enzymes such as the cytochromes P450.

The lovastatin nonaketide synthase catalysis of the intramolecular Diels-Alder reaction of a substrate analogue is reported

A solution NMR spectroscopic study of the cyanide-inhibited, substrate-bound complex of uniformly [SUP15]N-labeled human heme oxygenase, hHO, has led to characterization of the active site with respect to the nature and identity of strong hydrogen bonds and the occupation of ordered water molecules within both the hydrogen bonding network and an aromatic cluster on the distal side. {[SUP1]H-[SUP15]N}-HSQC spectra confirm the functionalities of several key donors in particularly robust H-bonds, and {[SUP1]H-[SUP15]N}HSQC-NOESY spectra lead to the identification of three additional robust H-bonds, as well as the detection of two more relatively strong H-bonds whose identities could not be established. The 3D NMR experiments provided only a modest, but important, extension of assignments because of the loss of key TOCSY cross-peaks due to the line broadening from a dynamic heterogeneity in the active site. Steady-state NOEs upon saturating the water signal locate nine ordered water molecules in the immediate vicinity of the H-bond donors, six of which are readily identified in the crystal structure. The additional three are positioned in available spaces to account for the observed NOEs. [SUP15]N-filtered steady-state NOEs upon saturating the water resonances and [SUP15]N-filtered NOESY spectra demonstrate significant negative NOEs between water molecules and the protons of five aromatic rings. Many of the NOEs can be rationalized by water molecules located in the crystal structure, but strong water NOEs, particularly to the rings of Phe47 and Trp96, demand the presence of at least an additional two immobilized water molecules near these rings. The H-bond network appears to function to order water molecules to provide stabilization for the hydroperoxy intermediate and to serve as a conduit to the active site for the nine protons required per HO turnover. [ABSTRACT FROM AUTHOR]

Solution 1H NMR is used to probe the environments of the donor protons of eight strong hydrogen bonds on the distal side of the heme substrate in the cyanide-inhibited, substrate-bound complex of human heme oxygenase, hHO. It is demonstrated that significant magnetization transfer from the bulk water signal to the eight labile protons does not result from chemical exchange, but from direct nuclear Overhauser effect due to the dipolar interaction of these labile protons with "ordered" water molecules. The enzyme labile proton to water proton distances are estimated at approximately 3 A. It is proposed that the role of the strong hydrogen-bonding network is to immobilize numerous water molecules which both stabilize the activated hydroperoxy species and funnel protons to the active site.