NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

Abstract

The role of nitric oxide (NO) in the host response to infection and in cellular signaling is well established. Enzymatic synthesis of NO is catalyzed by the nitric oxide synthases (NOSs), which convert Arg into NO and citrulline using co-substrates O2 and NADPH. Mammalian NOS contains a flavin reductase domain (FAD and FMN) and a catalytic heme oxygenase domain (P450-type heme and tetrahydrobiopterin). Bacterial NOSs, while much less studied, were previously identified as only containing the heme oxygenase domain of the more complex mammalian NOSs. We report here on the characterization of a NOS from Sorangium cellulosum (both fulllength, scNOS, and oxygenase domain, scNOSox). scNOS contains a catalytic, oxygenase domain similar to those found in the mammalian NOS and in other bacteria. Unlike the other bacterial NOSs reported to date, however, this protein contains a fused reductase domain. The scNOS reductase domain is unique for the entire NOS family because it utilizes a 2Fe2S cluster for electron transfer. scNOS catalytically produces NO and citrulline in the presence of either tetrahydrobiopterin or tetrahydrofolate. These results establish a bacterial electron transfer pathway used for biological NO synthesis as well as a unique flexibility in using different tetrahydropterin cofactors for this reaction.heme protein ͉ iron-sulfur cluster ͉ reductase ͉ tetrahydrobiopterin ͉ tetrahydrofolate N itric oxide (NO) is recognized as an important signaling molecule as well as a cytotoxin (1-3). A number of diseases are intimately tied to improper function of NO in humans (1-3). Given the importance of NO to human health, a wealth of studies has been performed to address questions regarding NO synthesis and regulation (4-9). Isolation and structural characterization of the proteins responsible for NO synthesis have led to important developments in understanding the molecular processes of NO formation (10-12). Three isoforms of nitric oxide synthase (NOS), iNOS, eNOS, and nNOS, have been characterized in mammals. These enzymes contain an oxygenase domain where the catalysis takes place and a reductase domain that is involved in electron transfer (4, 9). NOS is functional only as a homodimer, and requires the binding of a Ca 2ϩ -calmodulin (CaM) complex for electron transfer between the reductase and oxygenase domains. The reductase domain transfers electrons from NADPH (nicotinamide adenine dinucleotide phosphate) via FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) to the P450-type heme in the oxygenase domain (13). NOS contains an additional reduced pterin cofactor in the oxygenase domain [H 4 B, (6R)-tetrahydro-l-biopterin], which is required for NO formation and is involved in electron transfer processes during catalysis at the heme (14-16).NOS catalyzes the conversion of Arg into NO and citrulline using O 2 and NADPH involving two catalytic steps. In the first reaction, Arg is oxidized to N G -hydroxy-arginine (NHA). Nitric oxide is generated in the second half of the cycle, with the conversion...

The molecular mechanism of mammalian NO-synthases: A story of electrons and protons

“…In particular, the in silico analysis of numerous bacterial genomes has revealed the presence of NOS-like proteins in Streptomyces [226], Staphylococcus [227], Deinococcus [115], Bacillus [112,115,228,229]. Their crystallographic 3-D structure is extremely similar to that of the oxygenase domain of mammalian NOSs [112,167,228,[230][231][232], which seems to provide them with the necessary fundamental machinery to synthesize NO • [113,115,233,234]. However a significant release of NO • by bacterial NOSs both in vitro [115] and in vivo [235,236] remains to be demonstrated.…”

Influence of Heme-Thiolate in Shaping the Catalytic Properties of a Bacterial Nitric-oxide Synthase

“…BsNOS proteins were expressed and purified as described (34). Protein concentration was determined from the absorbance at 444 nm of the ferrous heme-CO complex, using an extinction coefficient of 76 mM Ϫ1 cm Ϫ1 (⌬⑀ 444 -500 nm ) (25). All proteins were purified to homogeneity (Ն95%) as assessed by SDS-PAGE FIGURE 2.…”

“…A number of bacterial NOS or NOS-like proteins have been isolated and characterized during the past 10 years . With the exception of the complete NOS present in Sorangium cellulosum (25), all of the bacterial NOSs characterized to date consist of a truncated NOSoxy domain that lacks a portion of the N-terminal region and has no attached reductase domain (30). Thus, NO synthesis by bacterial NOSs relies on their interaction with nondedicated reductases (3,4,20,22).…”

Nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation

“…S3). Interestingly, the only bacterial NOS that lack this tryptophan residue use an alternative N-terminal iron-sulfur cluster reductase domain in place of the typical C-terminal reductase domain (39).…”