Resonance Raman Investigation of Fe−N−O Structure of Nitrosylheme in Myoglobin and Its Mutants

Abstract

Resonance Raman spectra have been observed for NO adducts of wild-type (WT) sperm whale myoglobin (MbNO) and its H64G, H64L, L29W, V68W, and V68T mutants at neutral and acidic pH. Raman excitation in resonance with the Soret band enabled us to detect the Fe−NO stretching (νFe - NO), N−O stretching (νNO), and Fe−N−O bending (δFeNO) bands. The νFe - NO, δFeNO, and νNO bands of WT MbNO at neutral pH were observed at 560, 452, and 1613 cm-1, respectively, and substitution of the distal His64 to Gly or Leu caused an upshift of νNO to 1631−1635 cm-1 but no change in νFe - NO. This change in νNO is considered to be due to the removal of hydrogen bonding between His64 and bound NO. Conversely, substitution of Leu29 with tryptophan (L29W) altered νFe - NO but caused no change in νNO at neutral pH. This feature resembles that of MbO2 but distinctly differs from that of MbCO, for which the Fe−CO and C−O stretching frequencies have an inverse linear correlation. The change in νFe - NO for L29W−MbNO is probably caused by tilting of the Fe−N bond from the heme normal on account of steric hindrance from the large indole ring but would not be due to changes in the Fe−N−O bond angle. When pH is lowered to 4, MbNO adopts the five-coordinate structure due to cleavage of the Fe−His bond. Accordingly, the heme maker bands such as ν3 and ν10, shifted from 1500 and 1636 cm-1 at pH 7.4 to 1509 and 1646 cm-1 at pH 4 which are in agreement with those of a five-coordinate Fe−protoporphyrin−NO complex in detergent micelles at neutral pH. The νFe - NO and νNO bands of acidic MbNO were observed at 520 and 1668 cm-1 and exhibited no shift when the distal His was replaced by Gly or Leu. The latter observation supports previous X-ray crystallographic, infrared, and resonance Raman spectroscopic measurements which show that the distal histidine becomes protonated at pH 4 and swings out into the solvent away from the bound ligand.

Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase

“…However, the bands at 497 and 496 cm Ϫ1 were assigned to ␦(Fe-N-O) bending rather (Fe-NO) stretching (31) on the basis of IR studies of metal nitrosyl complexes, which indicated larger 15 NO isotope shifts for ␦(M-N-O) modes (6-15 cm Ϫ1 ) than for (M-NO) modes (1-6 cm Ϫ1 ) (38,39). Subsequent resonance Raman studies of nitrosyl hemes (19)(20)(21) have clearly shown that these ranges are applicable only to near-linear MNO units. Moreover, the pattern of isotope shifts observed for the 497 and 496 cm Ϫ1 bands in these complexes, i.e., larger isotope shift for 15 NO than N 18 O (31), is a characteristic of the (M-NO) modes of bent FeNO units in nitrosyl hemes (20).…”

“…Six-coordinate S ϭ 1͞2 {FeNO} 7 species in heme proteins have much stronger Fe-NO bonds, as evidenced by (Fe-NO) frequencies in the range 536-558 cm Ϫ1 (20,21,41), and much weaker N-O bonds, as evidenced by (N-O) frequencies in the range 1,555-1,624 cm Ϫ1 (20,21). Although no clear consensus has emerged concerning the most appropriate description of the electronic structure and bonding in S ϭ 1͞2 {FeNO} 7 species (18,32,42), the increase in Fe-NO bond strength and concomitant decrease in the N-O bond strength, compared with S ϭ 3͞2 {FeNO} 7 species, appear to be a direct consequence of increased -donation into the empty d z2 orbital that results from lowering the Fe spin state from high to low or intermediate spin.…”

“…First, in the absence of a specific enhancement mechanism, bending modes generally exhibit weaker resonance enhancement than stretching modes and hence are weaker in resonance Raman spectra. This general observation is borne out by resonance Raman studies of nitrosyl hemes in which the (Fe-NO) stretching and ␦(Fe-N-O) bending modes have been rigorously assigned based on 15 (19)(20)(21). In addition to the (Fe-NO) and (N-O) stretching modes, vibrational modes associated with the Fe-S(Cys) unit are also weakly enhanced in resonance with absorption band at 21,050 cm Ϫ1 (475 nm).…”

“…However, these intermediates are short lived and difficult to study experimentally. Nitric oxide (NO) has been extensively used as a substrate analog of molecular oxygen to form stable nitrosyl derivatives that provide insight into oxygen transport and activation intermediates in many heme (18)(19)(20)(21) and nonheme (22)(23)(24)(25)(26)(27)(28)(29) iron enzymes. Hence, we report here the formation and spectroscopic characterization of a stable NO-bound derivative of the reduced 1Fe-SOR from P. furiosus using the combination of EPR, UV-visible absorption, and variable-temperature, variable-field magnetic CD (VTVH MCD), resonance Raman, and Fourier transform IR (FTIR) spectroscopies.…”

Complex Formation of Cytochrome P450cam with Putidaredoxin

“…Recently, Tomita et al (19) and Vogel et al (20) performed resonance Raman studies on ferrous NO adducts of myoglobin and heme model compounds and showed that the vibrational properties of the Fe-NO unit are distinctly different from those of the Fe-CO unit. For the CO ligand, the two main factors that affect the Fe-CO and C-O frequencies are: 1) the polarity of distal residues in the heme pocket and their proximity to the CO, and 2) the nature of the axial ligand trans to the CO (16,17,21,22).…”