Resonance Raman Studies of Cytochrome P450bm3 and Its Complexes with Exogenous Ligands

Abstract

Resonance Raman spectra are reported for both the heme domain and holoenzyme of cytochrome P450BM3 in the resting state and for the ferric NO, ferrous CO, and ferrous NO adducts in the absence and presence of the substrate, palmitate. Comparison of the spectrum of the palmitate-bound form of the heme domain with that of the holoenzyme indicates that the presence of the flavin reductase domain alters the structure of the heme domain in such a way that water accessibility to the distal pocket is greater for the holoenzyme, a result that is consistent with analogous studies of cytochrome P450cam. The data for the exogenous ligand adducts are compared to those previously reported for corresponding derivatives of cytochrome P450cam and document significant and important differences for the two proteins. Specifically, while the binding of substrate induces relatively dramatic changes in the nu(Fe-XY) modes of the ferrous CO, ferric NO, and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the corresponding derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for distortion of the Fe-XY fragment, even in the absence of substrate. This apparent distortion, which is nonexistent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of the Fe-XY fragment with the F87 phenylalanine side chain. This residue is known to lie very close to the heme iron in the substrate-free derivative of cytochrome P450BM3 and has been suggested to prevent hydroxylation of the terminal, omega, position of long-chain fatty acids.

Structural Diversities of Active Site in Clinical Azole-bound Forms between Sterol 14α-Demethylases (CYP51s) from Human and Mycobacterium tuberculosis

“…The same spectral pattern was observed for HuCYP51. These observed frequencies of the marker lines in the two CYP51s were almost identical to those in CYP101, CYP102, and CYP121, which indicates that the heme of CYP51s are planar as found for other CYP enzymes (37)(38)(39).…”

Role of Arginine Guanidinium Moiety in Nitric-oxide Synthase Mechanism of Oxygen Activation

“…On the other hand, we and others have shown that the nature of the guanidine substrate can significantly modify the oxidation chemistry, along with the resulting reaction intermediates and products (41,42,71,72). These considerations led us to address, in this work, the actual role of the guanidinium proton in the mechanism of NOS by analyzing the interactions between the heme active site and the guanidinium moiety of L (41, 55-57, 59, 69, 70) and cytochromes P450 (44,68,(73)(74)(75)(76). In addition to bond angles, the Fe-CO vibrational modes are very sensitive to the electrostatic and polar properties of the heme distal pocket because of changes in the back donation from the iron d* orbital to the empty * CO orbital (44,45).…”

Interactions between substrates and the haem-bound nitric oxide of ferric and ferrous bacterial nitric oxide synthases

“…However, as the ν N−O mode is usually not resonance enhanced with Soret excitation [38], we could not localize this mode in the high-frequency region of the FeIII-NO spectra using isotope substitution (results not shown). [24,40], iNOSox [24,41], P450 nor [42] and P450 BM3 [43]. The line at 539 cm −1 ( 4 cm −1 ) of saNOS was thus assigned to the ν Fe−NO mode.…”

“…The frequencies of the ν Fe−NO modes of saNOS (539 cm −1 ) and bsNOS (540 cm −1 ) in the absence of substrate and H 4 B were nearly identical, indicating that both proteins had very similar haem environments. These frequencies were compared with those of other haem proteins and were found to most resemble those of nNOSox (535 cm −1 ) and iNOSox (537 cm −1 ) rather than P450 cam (528 cm −1 ; [39]) and P450 BM3 (526 cm −1 ; [43]), indicating a similar environment at the active sites of bacterial and mammalian NOSs ( Table 1). The frequencies of the ν Fe−NO modes of saNOS and bsNOS were insensitive to the addition of the pterins H 4 B and THF (Table 1).…”