Resonance Raman study of Bacillus subtilis NO synthase-like protein: similarities and differences with mammalian NO synthases.
Bacterial NO synthase (NOS)-like proteins such as that from Bacillus subtilis (bsNOS) share a high degree of structural homology with the oxygenase domain of mammalian NOSs (mNOSs), but biochemical studies have yet failed to establish that they are specifically capable of producing NO. To better understand the actual function and role of bacterial NOSs, the structure and environment of bsNOS heme were examined with resonance Raman (RR) and ATR-FTIR spectroscopies. We analyzed the structural effects of l-arginine (Arg) and tetrahydrobiopterin (H(4)B) binding on several key complexes (ferric, ferrous, ferrous-CO, and ferric-NO) and characterized the bonding properties of the proximal cysteine ligand. While our study fully confirms the similarity between bsNOS and mNOS heme pocket structures, our results also highlight important differences. (i) Contrary to other NOSs, resting native ferric bsNOS exhibits an exclusive five-coordinate high-spin iron status. (ii) The nu(Fe)(-)(CO) and nu(CO) mode frequencies of the bsNOS Fe(II)CO complexes indicate a weaker electrostatic interaction between Arg and CO. (iii) bsNOS is characterized by a stronger Fe-S bond (nu(Fe)(-)(S) = 342 cm(-)(1)), a lower nu(4) frequency, and a negative shift in the nu(Fe)(-)(CO)/nu(CO) correlation. (iv) The effects of H(4)B on bsNOS heme structure are minor compared to the ones reported on mNOS. These results suggest distinct distal heme environments between mNOS and bsNOS, greater electron-donation properties of bsNOS cysteine proximal ligand, and the absence of a significant influence of H(4)B on bsNOS heme properties. These subtle structural differences may reflect changes in the chemistry and physiological role of bacterial NOSs.
Santolini
Jérôme
J
Département de Biologie Joliot-Curie, CEA Saclay, 91191 Gif sur Yvette Cedex, France. jerome.santolini@cea.fr
Roman
Miruna
M
Stuehr
Dennis J
DJ
Mattioli
Tony A
TA
D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology*; D002248:Carbon Monoxide / Q000737:chemistry; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D051379:Mice; D009569:Nitric Oxide / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014732:Vibration
D005290:Ferric Compounds; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D019001:Nitric Oxide Synthase