Spectroscopic Characterization of the Nitrophorins

Optical Spectroscopy of Nitrophorins in the Absence and Presence of NO and Other Ligands 

The optical spectra of nitrophorins in the absence of added ligands show Soret band maxima at 403-404 nm. On binding NO, the Soret band shifts to 419-420 nm in NPl and NP4, and 421-423 nm in NP2 and NP3 (46, 48-50). Example spectra are shown in Fig. 5a. The direction of the Soret band shift identifies the oxidation state of the heme iron as being Fe(III) (51, 52). The a and (3 bands of the NO complexes are located near 570 and 535 nm, respectively. The histamine complexes have Soret maxima at 410-412 nm and broad a, (3 maxima between 580 

The optical spectra of the nitrophorins in the oxidized (Fe(III)) and reduced (Fe(II)) states have been measured by spectroelectrochemical techniques, as shown for NP3 and NP3-NO at pH 7.5 in Figs. 6a and 6b, respectively. As reported previously (49, SO), there is only a 3 nm shift in 

For NP2 and NP3 at pH 7.5, the shift in Soret band positions of the NO complexes for the two oxidation states is somewhat larger—8-10 nm, from 421-423 to 413 nm for the Fe(III) and Fe(II) complexes, respectively (50). However, in contrast to NPl-NO (49) and NP4-NO (50), at pH 5.5 NP2-NO and NP3-NO show very different spectral shifts upon electrochemical reduction, as shown in Fig. 6c for NP3-NO. The Soret band shifts to 395 nm, and both the wavelength maximum and shape of the Soret band are typical of five-coordinate heme-NO centers, including guanylyl cyclase, upon binding NO (53, 54). The reduced forms of both NP2-NO and NP3-NO exhibit similar pH dependence of the absorption spectra, whereas NPl-NO and NP4-NO do not show any pH dependence of their absorption spectra over the pH range 5.5-7.5 (50). 

The optical spectral data for the NP2 -NO and NP3 -NO complexes at pH 5.5 suggest that there may be a difference in Fe-N bond strength of the proximal histidine ligand for these two proteins that is only apparent when the metal is reduced to Fe(II). Thus, the kinetic differences in NO release behavior of the NP1,4 group and the NP2,3 group, discussed later, could in part be related to differences in heme-histidine bond strength. If so, then it must be concluded that a weakened Fe-N bond strength produces a more stable Fe-NO complex, since the Ki values for NP2,3-NO are much smaller than for NPl,4-NO (50) (see Section IV). 

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