Stability of the heme environment of the nitric oxide synthase from Staphylococcus aureus in the absence of pterin cofactor.
We have used resonance Raman spectroscopy to probe the heme environment of a recently discovered NOS from the pathogenic bacterium Staphylococcus aureus, named SANOS. We detect two forms of the CO complex in the absence of L-arginine, with nu(Fe-CO) at 482 and 497 cm(-1) and nu(C-O) at 1949 and 1930 cm(-1), respectively. Similarly to mammalian NOS, the binding of L-arginine to SANOS caused the formation of a single CO complex with nu(Fe-CO) and nu(C-O) frequencies at 504 and 1,917 cm(-1), respectively, indicating that L-arginine induced an electrostatic/steric effect on the CO molecule. The addition of pterins to CO-bound SANOS modified the resonance Raman spectra only when they were added in combination with L-arginine. We found that (6R) 5,6,7,8 tetra-hydro-L-biopterin and tetrahydrofolate were not required for the stability of the reduced protein, which is 5-coordinate, and of the CO complex, which does not change with time to a form with a Soret band at 420 nm that is indicative of a change of the heme proximal coordination. Since SANOS is stable in the absence of added pterin, it suggests that the role of the pterin cofactor in the bacterial NOS may be limited to electron/proton transfer required for catalysis and may not involve maintaining the structural integrity of the protein as is the case for mammalian NOS.
Chartier
François J M
FJ
Department of Biochemistry and Microbiology, Laval University, Quebec City, Quebec G2K 7P4, Canada.
Couture
Manon
M
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D002248:Carbon Monoxide; D004583:Electrons; D006418:Heme / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D011622:Pterins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013211:Staphylococcus aureus / Q000201:enzymology*; D055672:Static Electricity; D013763:Tetrahydrofolates / Q000737:chemistry
D011522:Protons; D011622:Pterins; D013763:Tetrahydrofolates; D006418:Heme; C030371:5,6,7,8-tetrahydrofolic acid; D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase