ABSTRACT
Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme-HO-1, whereas the reduction in heme-HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme-HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme-HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme-HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme-HO-1 in solution. We conclude that the "open-close" transition of CPR is indispensable for electron transfer from CPR to heme-HO-1.
ABSTRACT
Soybean seeds contain phytochemicals such as polyamines and isoflavones, which have been identified as functional components mediating health benefits in association with the consumption of soy foods. While a clear picture of the spatial distribution of these components within the seed is lacking, such information would be important to enhance or reduce their concentration in respective foods through processing. Thus, the objective of the present study was to visualize the most relevant components with respect to their distribution in soybean seeds. Mature soybean seeds were subject to atmospheric-pressure scanning-microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) combined with a Fourier-transform orbital trapping mass spectrometer to generate high-resolution chemical images of phytochemical distribution. Based on seed cross sections, differential distributions of functional components were found between soybean cotyledon and germ (shoot, hypocotyl, root) regions. Spermidine and spermine were present in higher concentrations in the germ rather than in cotyledons with highest concentrations in root and shoot meristem tissues. Differential concentrations of spermidine and other components between the germ and cotyledon regions were confirmed by seed fractioning. In contrast to polyamines spermidine and spermine, the different types of daidzein, glycitein, and genistein isoflavones were all visualized in root parenchyma tissue exclusively. Overall, mass spectrometry imaging of soybean seeds revealed clear insights into the differential distribution of functional phytochemicals. Based on their distribution and depending on specific needs, spermidine and isoflavones can either be enriched or reduced during food processing by separating cotyledon and germ fractions.
