Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase

Abstract

Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local probes of the active site of iNOS. Characteristic changes were observed in the spectra of the heme-bound ligands upon binding of the cofactors. Unlike photolyzed CO, which becomes trapped in well-defined orientations, as indicated by sharp photoproduct bands, photoproduct bands of NO photodissociated from the ferric heme iron were not visible, indicating that NO does not reside in the protein interior in a well-defined location or orientation. This may be favorable for NO release from the enzyme during catalysis because it reduces self-inhibition. Moreover, we used temperature derivative spectroscopy (TDS) with FTIR monitoring to explore the dynamics of NO and carbon monoxide (CO) inside iNOS after photodissociation at cryogenic temperatures. Only a single kinetic photoproduct state was revealed, but no secondary docking sites as in hemoglobins. Interestingly, we observed that intense illumination of six-coordinate ferrous iNOS oxy-NO ruptures the bond between the heme iron and the proximal thiolate to yield five-coordinate ferric iNOS oxy-NO, demonstrating the strong trans effect of the heme-bound NO.

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…The experimental results derived from the data of the 11 new model complexes are in excellent agreement with DFT calculations on Cyt. P450 (heme-thiolate) enzyme mimics, , and the collective experimental results available for the NO adducts of different heme-thiolate enzymes. ,− With findings (i)–(iv) now firmly established, based on experimental data, it is concluded that the differences in Fe–NO and N–O stretching frequencies between different heme-thiolate ls-{FeNO} 6 protein complexes are largely due to variations in the number and strength of hydrogen bonds to their respective cysteinate sulfurs. Figure compares the data points of the 11 heme-thiolate ls-{FeNO} 6 model complexes with those reported for ls-{FeNO} 6 adducts in a number of heme-thiolate proteins (see also Table ), and shows the two correlation lines established in the model complex studies. , The slopes obtained from two different linear fits of the data points in Figure (1.20 and 1.61) establish a direct correlation of the Fe–NO and N–O stretching frequencies, and hence bond strengths, with the donor strength of the axial thiolate ligand.…”

“…This intermediate thiolate donor strength in Cyt. P450nor can also be seen by comparing its N–O stretching frequency to those of the ls-{FeNO} 6 complexes of NOS and chloroperoxidase (CPO; observed at 1870 and 1868 cm –1 , respectively), − which are known to have weaker thiolate donors than Cyt. P450s due to the presence of stronger proximal hydrogen bonds to their cysteinate ligands.…”

Kinetic Study of Ligand Binding and Conformational Changes in Inducible Nitric Oxide Synthase

“…We have shown earlier that iNOS oxy does not have any additional ligand docking sites beyond the primary site close to the heme iron, where a ligand can transiently be stored. 12 The remarkable difference between CO rebinding in P420 and P450 thus points to pronounced structural differences in the ligand binding pockets between the two species that accompany the protonation of the axial ligand Cys194 and affect the CO migration pathway.…”

“…We have previously investigated ligand and substrate binding in iNOS oxy by using Fourier transform infrared (FTIR) spectroscopy of the CO stretching vibration in the midinfrared. 12 These absorption bands are fine-tuned by electrostatic interactions with the environment, which makes CO a superb local probe of protein structure and dynamics in the interior of heme proteins. In a freshly prepared P450 iNOS oxy sample at 4 K, the CO stretching bands are centered on 1921, 1945, and 1959 cm −1 .…”

“…In addition, we frequently employ infrared spectroscopy of the CO stretching absorption as a superb complementary marker for probing the active site. 8,10,12 Here, we have analyzed the effects of cofactor and substrate on CO rebinding to the heme iron in a mammalian inducible nitric oxide synthase (iNOS). The enzyme is expressed in macrophages and up-regulated upon inflammatory and immunologic stimulation so as to generate highly reactive nitric oxide (NO) to protect against microbial pathogens.…”