High pressure fourier transform infrared (FT-IR) spectroscopic studies on inducible nitric oxide (NO) synthase active site: a comparison to cytochrome p450CAM.
High pressure Fourier transform infrared (FT-IR) spectroscopy is performed for the first time to analyse the active site of inducible nitric oxide synthase (iNOSox) using the carbon monoxide (CO) heme iron ligand stretch mode (nuCO) as spectroscopic probe. A membrane-driven sapphire anvil high-pressure cell is used. Three major conformational substates exist in substrate-free iNOSox which are characterized by nuCO at approximately 1936, 1945 and 1952 cm(-1). High pressure favors the 1936 cm(-1) substate with a volume difference to the 1945 substate of approximately -21 cm3/mol. The pressure induced cytochrome P420 formation with a reaction volume of approximately -80 cm3/mol is observed. Arginine binding produces a very low nuCO at approximately 1905 cm(-1) caused by the H-bond from the substrate to CO. nuCO for the substates in the substrate-free and arginine-bound proteins shift linearly with pressure which is qualitatively similar to the observation on cytochrome P450cam. The slightly smaller positive slope of the shift in substrate-free iNOSox compared to substrate-free P450cam is interpreted as a slightly lesser compressible heme pocket. In contrast, the significant slower negative slope for arginine-bound iNOSox compared to camphor-bound P450cam results from the different kind of interactions to the CO ligand (electrostatic interaction in P450cam, H-bond in iNOSox).
Jung
C
C
Max-Delbrück-Center for Molecular Medicine, Protein Dynamics Laboratory, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany. cjung@mdc-berlin.de
Ghosh
D K
DK
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry; D002248:Carbon Monoxide / Q000737:chemistry; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II; D044367:Phase Transition; D011312:Pressure; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D055672:Static Electricity
D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D019475:Camphor 5-Monooxygenase