Resonance Raman structural characterization of cytochrome P-450cam and lactoperoxidase
Abstract
The resonance Raman spectra of nitric oxide adducts of ferric and ferrous cytochrome P450cam as well as the oxygenated cytochrome P450cam, in the presence of various substrate analogues, are studied. For the nitric oxide derivative of camphor-bound ferric enzyme, $\nu$(Fe$\sp{\rm III}$-NO), $\delta$(Fe$\sp{\rm III}$-N-O) modes, are detected at 522 and 546 cm$\sp{-1}$, respectively. They shift to 520 and 533 cm$\sp{-1}$ upon substitution by $\sp{15}$N$\sp{16}$O. The strong line at 522 cm$\sp{-1}$ shifts to 528 cm$\sp{-1}$ when the substrate is removed from the active site. As the substrate size increases, a band of medium intensity, which is tentatively assigned as $\nu$(Fe-S), increases from 349 (substrate-free) to 359 cm$\sp{-1}$ (adamantanone). The three expected normal modes of the nitric oxide adduct of ferrous cytochrome P450cam are detected at 1591, 554 and 446 cm$\sp{-1}$, which are assigned to the $\nu$(N-O), $\nu$(Fe$\sp{\rm II}$-NO) and $\delta$(Fe$\sp{\rm II}$-NO) respectively, based on a normal mode analysis. The very strong band at 554 cm$\sp{-1}$ shifts to 539 ($\sp{15}$N$\sp{16}$O), 552 ($\sp{14}$N$\sp{18}$O) and 538 cm$\sp{-1}$ ($\sp{15}$N$\sp{18}$O) as the mass of NO increases, while the one at 446 cm$\sp{-1}$ shows a monotonous downshift to 442 ($\sp{15}$N$\sp{16}$O), 440 ($\sp{14}$N$\sp{18}$O) and 437 cm$\sp{-1}$ ($\sp{15}$N$\sp{18}$O). The substrate sensitivity of these two bands suggests that only camphor can exert steric pressure on the bound NO fragment. Both the $\nu$(O-O) and $\nu$(Fe-O$\sb2$) modes are simultaneously observed and identified (by using $\sp{16}$O$\sb2$/$\sp{18}$O$\sb2$ isotopic substitution technique) at 1140 and 541 cm$\sp{-1}$, respectively for camphor-bound oxygenated cytochrome P450cam. When camphor is replaced with adamantanone, two lines at 1139 and 1147 cm$\sp{-1}$ are observed for the $\nu$(O-O) mode, while no significant change in heme core structure is discerned. Lactoperoxidase compound III (LPO-III) is characterized by resonance Raman spectroscopy with both the Soret and Q band excitations. The Fe-O$\sb2$ stretching vibration (identified at 531 cm$\sp{-1}$ for the $\sp{16}$O$\sb2$ adduct and 513 cm$\sp{-1}$ for the $\sp{18}$O$\sb2$ derivative) is the lowest yet reported for an oxygenated heme protein. The anomalous isotopic shift of $\nu$(Fe-O$\sb2$) mode is attributed to vibrational coupling of Fe-$\sp{18}$O$\sb2$ stretching mode with a heme mode located at 508 cm$\sp{-1}$. The study of the $\nu$(Fe-O$\sb2$) mode of LPO-III, prepared by different combinations of isotopically-labeled peroxides, establishes unambiguously that the ferryl oxygen atom of LPO-II is displaced and the dioxygen fragment of LPO-III is derived from the hydrogen peroxide.
Recommended Citation
Hu, Songzhou, "Resonance Raman structural characterization of cytochrome P-450cam and lactoperoxidase" (1991). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI9133796.
https://epublications.marquette.edu/dissertations/AAI9133796