Comparative Functioning of Dihydro- and Tetrahydropterins in Supporting Electron Transfer, Catalysis, and Subunit Dimerization in Inducible Nitric Oxide Synthase

Abstract

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

“…These findings reveal that the stabilizing effects of H % Bip are probably independent of the composition of the C-6 pterin side chain. Similar conclusions were made by Presta et al [54] for NOS-II on dimer reassembly by various analogues of pterin after urea-induced monomerization. In the present study, PHS-32 was less effective than H % Bip at stabilizing dimeric NOS-I and this might be related to the inability of PHS-32 to support enzyme catalysis and allosterically regulate -arginine binding.…”

Anti-pterins as Tools to Characterize the Function of Tetrahydrobiopterin in NO Synthase

“…Purified NOS-I exists as a stable homodimer (27), and monomerization was only described to occur under denaturing conditions, i.e. in the presence of 4% SDS (16) or 5 M urea (12). However, we observed monomerization to occur also during L-arginine turnover, i.e.…”

“…For example, H 4 Bip acts catalytically as the sole oxygen acceptor for the aromatic amino acid hydroxylases (1); for NOS, this role is already fulfilled by the cytochrome P 450 -type heme (10,11). In a recent study (12) it was shown that heme iron reduction can take place independently of the pterin ring oxidation state. Although this would indicate a requirement for fully reduced pterin at a reaction step beyond heme iron reduction, recent evidence (13) suggests that the reduction of the oxyferroheme complex may be the main function of H 4 Bip in NOS catalysis.…”

“…However, in the case of recombinant NOS-I and NOS-III, dimer formation appears to be pterin-independent and regulated solely by the intracellular availability of heme (19,20). Related structural effects of H 4 Bip that have been described include an increase in temperature stability of NOS-I dimers in the presence of the protein denaturant SDS (16) and reassembly of urea-dissociated dimers (12). Nevertheless, the function of H 4 Bip in NOS catalysis has remained unclear.…”

Kinetics of acid-induced degradation of tetra- and dihydrobiopterin in relation to their relevance as biomarkers of endothelial function

“…BH 4 is essential for the production of nitric oxide, an endothelial vasorelaxing factor, by NOS and may hence serve as a biomarker of endothelial dysfunction. While BH 2 may bind to NOS just like BH 4 (Vásquez-Vivar et al, 2002;Crabtree et al, 2008), it does not support the formation of nitric oxide (Presta et al, 1998) and instead superoxide is formed (Vásquez-Vivar et al, 2002). Studies have suggested that the ratio of BH 4 to BH 2 correlates better with endothelial function than the absolute concentration of BH 4 (Noguchi et al, 2011;Crabtree et al, 2008;Takeda et al, 2009;Kar & Kavdia, 2011).…”