2FLQ

Crystal Structure of Nitric Oxide Synthase from Geobacillus Stearothermophilus (ATCC 12980) complexed with L-arginine

Summary for 2FLQ

• Entry DOI: 10.2210/pdb2flq/pdb
• Descriptor: Nitric Oxide Synthase, ARGININE, PROTOPORPHYRIN IX CONTAINING FE, ... (4 entities in total)
• Functional Keywords: nitric oxide synthase, geobacillus stearothermophilus, thermostable enzyme, oxidoreductase
• Biological source: Geobacillus stearothermophilus
• Total number of polymer chains: 2
• Total formula weight: 88944.35

Authors

Sudhamsu, J.,Crane, B.R. (deposition date: 2006-01-06, release date: 2006-01-31, Last modification date: 2023-08-30)

Primary citation

Sudhamsu, J.,Crane, B.R.
Structure and Reactivity of a Thermostable Prokaryotic Nitric-oxide Synthase That Forms a Long-lived Oxy-Heme Complex.
J.Biol.Chem., 281:9623-9632, 2006

PubMed Abstract: In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates l-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H(4)B. The crystal structure of gsNOS at 3.2 AA of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 degrees C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.

PubMed: 16407211
DOI: 10.1074/jbc.M510062200

Experimental method

X-RAY DIFFRACTION (3.2 Å)