Resonance Raman Detection of the Fe−S Bond in Endothelial Nitric Oxide Synthase^,

Abstract

We report the first low-frequency resonance Raman spectra of ferric endothelial nitric oxide synthase (eNOS) holoenzyme, including the frequency of the Fe-S vibration in the presence of the substrate L-arginine. This is the first direct measurement of the strength of the Fe-S bond in NOS. The Fe-S vibration is observed at 338 cm(-1) with excitation at 363.8 nm. The assignment of this band to the Fe-S stretching vibration was confirmed by the observation of isotopic shifts in eNOS reconstituted with 54Fe- and 57Fe-labeled hemin. Furthermore, the frequency of this vibration is close to those observed in cytochrome P450(cam) and chloroperoxidase (CPO). The frequency of this vibration is lower in eNOS than in P450(cam) and CPO, which can be explained by differences in hydrogen bonding to the proximal cysteine heme ligand. On addition of substrate to eNOS, we also observe several low-frequency vibrations, which are associated with the heme pyrrole groups. The enhancement of these vibrations suggests that substrate binding results in protein-mediated changes of the heme geometry, which may provide the protein with an additional tuning element for the redox potential of the heme iron. The implications of our findings for the function of eNOS will be discussed by comparison with P450(cam) and model compounds.

Heme Distortion Modulated by Ligand-Protein Interactions in Inducible Nitric-oxide Synthase

“…The low frequency region of the spectrum was not examined in this study. Nonetheless, in a study of eNOS, changes in certain low frequency modes were identified upon the addition of L-Arg and were interpreted as an indication of a protein structural change (27).…”

“…The Fe-S stretching mode of the proximal bond was identified at 338 cm Ϫ1 by Schelvis et al (27) in the resonance Raman spectrum with near-UV excitation. The Fe-S stretching frequency is lower than that in cytochrome P-450s, indicating a weaker Fe-S bond that may be important for the catalytic function of NOS.…”

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

“…S1B). Diagnostic heme porphyrin modes were observed around 1372 cm Ϫ1 ( 4 ), 1487 cm Ϫ1 ( 3 ), 1562 cm Ϫ1 ( 2 ), and 1625 cm Ϫ1 ( vinyl ) (supplemental Table S3), which are all characteristic of a sole population of iNOSoxy in the Fe III five-coordinated (5c) high spin state (56,58,(65)(66)(67). In the same way, the RR spectra of iNOSoxy S2) (55-57, 59, 66, 67).…”

Unique Heme Environmental Structures in Heme-regulated Proteins Using Heme as the Signaling Molecule

“…44 As illustrated in Figure 1, the resonance Raman line at 333 cm ¹1 clearly showed an isotopic shift associated with the heme iron ( 54 Fe and 56 Fe), which corresponds to that of the FeS stretching mode of other Cysligated proteins 45 and the calculated value, 46 confirming heme binding to Cys in heme-bound Irr. The line at 333 cm ¹1 was not detected in the mutant with a mutation at 29 Cys in HRM, indicating that this line originated from the bond between the heme iron and 29 Cys in HRM.…”

“…47 The lowest frequencies for the FeS stretching mode were reported for NPAS2 (334 cm ¹1 ) 48 and eNOS (338 cm ¹1 ) 45 in which the basicity of the axial thiolate is lower due to a weakened hydrogen bond between the sulfur atom of the thiolate and the nitrogen atoms of the amide groups in the main chain. The lower FeS stretching mode in heme-bound Irr than that of eNOS implies that 29 Cys in the HRM of Irr has no hydrogen bonds with the amide groups in the main chain to stabilize the FeS bond, as illustrated in Figure 2.…”