Thermodynamic and kinetic analysis of the nitrosyl, carbonyl, and dioxy heme complexes of neuronal nitric-oxide synthase. The roles of substrate and tetrahydrobiopterin in oxygen activation.
ABSTRACT Mammalian NO synthases catalyze the monooxygenation of L-arginine (L-Arg) to N-hydroxyarginine (NOHA) and the subsequent monooxygenation of this to NO and citrulline. Both steps proceed via formation of an oxyferrous heme complex and may ultimately lead to a ferrous NO complex, from which NO must be released. Electrochemical reduction of NO-bound neuronal nitricoxide synthase (nNOS) oxygenase domain was used to form the ferrous heme NO complex, which was found to be stable only in the presence of low NO concentrations, due to catalytic degradation of NO at the nNOS heme site. The reduction potential for the heme-NO complex was approximately -140 mV, which shifted to 0 mV in the presence of either L-Arg or NOHA. This indicates that the complex is stabilized by 14 kJ mol(-1) in the presence of substrate, consistent with a strong H-bonding interaction between NO and the guanidino group. Neither substrate influenced the reduction potential of the ferrous heme CO complex, however. Both L-Arg and NOHA appear to interact with bound NO in a similar way, indicating that both bind as guanidinium ions. The dissociation constant for NO bound to ferrous heme in the presence of l-Arg was determined electrochemically to be 0.17 nM, and the rate of dissociation was estimated to be 10(-4) s(-1), which is much slower than the rate of catalysis. Stopped-flow kinetic analysis of oxyferrous formation and decay showed that both l-Arg and NOHA also stabilize the ferrous heme dioxy complex, resulting in a 100-fold decrease in its rate of decay. Electron transfer from the active-site cofactor tetrahydrobiopterin (H4B) has been proposed to trigger the monoxygenation process. Consistent with this, substitution by the analogue/inhibitor 4-amino-H4B stabilized the oxyferrous complex by a further two orders of magnitude. H4B is required, therefore, to break down both the oxyferrousand ferrous nitrosyl complexes of nNOS during catalysis. The energetics of these processes necessitates an electron donor/acceptor operating within a specific reduction potential range, defining the role of H4B.
- SourceAvailable from: Mohammad Haque[show abstract] [hide abstract]
ABSTRACT: NO synthase enzymes (NOS) support unique single-electron transitions of a bound H(4)B cofactor during catalysis. Previous studies showed that both the pterin structure and surrounding protein residues impact H(4)B redox function during catalysis. A conserved Arg residue (Arg375 in iNOS) forms hydrogen bonds with the H(4)B ring. In order to understand the role of this residue in modulating the function of H(4)B and overall NO synthesis of the enzyme, we generated and characterized three mutants R375D, R375K and R375N of the oxygenase domain of inducible NOS (iNOSoxy). The mutations affected the dimer stability of iNOSoxy and its binding affinity toward substrates and H(4)B to varying degrees. Optical spectra of the ferric, ferrous, ferrous dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to the wild-type. However, mutants displayed somewhat lower heme midpoint potentials and faster ferrous heme-NO complex reactivity with O(2). Unlike the wild-type protein, mutants could not oxidize NOHA to nitrite in a H(2)O(2)-driven reaction. Mutation could potentially change the ferrous dioxy decay rate, H(4)B radical formation rate, and the amount of the Arg hydroxylation during single turnover Arg hydroxylation reaction. All mutants were able to form heterodimers with the iNOS G450A full-length protein and displayed lower NO synthesis activities and uncoupled NADPH consumption. We conclude that the conserved residue Arg375 (1) regulates the tempo and extent of the electron transfer between H(4)B and ferrous dioxy species and (2) controls the reactivity of the heme-based oxidant formed after electron transfer from H(4)B during steady state NO synthesis and H(2)O(2)-driven NOHA oxidation. Thus, Arg375 modulates the redox function of H(4)B and is important in controlling the catalytic function of NOS enzymes.Journal of inorganic biochemistry 11/2011; 108:203-15. · 3.25 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The vital signalling molecule NO is produced by mammalian NOS (nitric oxide synthase) enzymes in two steps. L-arginine is converted into NOHA (Nω-hydroxy-L-arginine), which is converted into NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor BH4 (tetrahydrobiopterin) activates dioxygen at the haem site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochrome P450, a haem-iron oxo species may be formed, or direct reaction between a haem-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. In the present paper we analyse the two reaction steps using the G586S mutant of nNOS (neuronal NOS), which introduces an additional hydrogen bond in the active site and provides an additional proton source. In the mutant enzyme, BH4 activates dioxygen as in the wild-type enzyme, but an interesting intermediate haem species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-arginine) indicating that the extra hydrogen bond enables it to discriminate between the two mono-oxygenation steps. This implies that the two steps follow different chemical mechanisms.Biochemical Journal 02/2012; 443(2):505-14. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Nitric oxide synthase (NOS), a flavo-hemoprotein, tightly regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a key signaling molecule for vasodilatation and neurotransmission at low concentrations, and also as a defensive cytotoxin at higher concentrations. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their key biological functions by tight regulation of interdomain electron transfer (IET) process via interdomain interactions. In particular, the FMN-heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O(2) activation at the catalytic heme site. Compelling evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS through a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the domains. Another exciting recent development in NOS enzymology is the discovery of importance of the the FMN domain motions in modulating reactivity and structure of the catalytic heme active site (in addition to the primary role of controlling the IET processes). In the absence of a structure of full-length NOS, an integrated approach of spectroscopic (e.g. pulsed EPR, MCD, resonance Raman), rapid kinetics (laser flash photolysis and stopped flow) and mutagenesis methods is critical to unravel the molecular details of the interdomain FMN/heme interactions. This is to investigate the roles of dynamic conformational changes of the FMN domain and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in understanding of mechanisms of the NOS regulation that are driven by the combined approach are the focuses of this review. An improved understanding of the role of interdomain FMN/heme interaction and CaM binding may serve as the basis for the design of new selective inhibitors of NOS isoforms.Coordination Chemistry Reviews 02/2012; 256(3-4):393-411. · 11.02 Impact Factor