Benjamin Hemmens

Biosynthesis and action of nitric oxide in mammalian cells

Nitric oxide (NO) can act as a vasorelaxant, a modulator of neurotransmission and a defence against pathogens. However, under certain conditions, NO can also have damaging effects to cells. Whether NO is useful or harmful depends on its chemical fate, and on the rate and location of its production. Here, we discuss progress in NO chemistry and the enzymology of NO synthases, and we will also attempt to explain its actions in the cardiovascular, nervous and immune systems.

NITRIC OXIDE: CHEMICAL PUZZLES POSED BY A BIOLOGICAL MESSENGER

Recent years have seen a massive growth of interest in the biological effects of nitric oxide (NO): It acts as a signaling molecule in blood vessels and the brain, and as a defense against pathogens in the immune system. Discussed are the chemical events underlying the physiology of this versatile little molecule.

Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) are homodimeric proteins and can form an intersubunit Zn(4S) cluster. We have measured zinc bound to NOS purified from pig brain (0.6 mol/mol of NOS) and baculovirus-expressed rat neuronal NOS (nNOS) (0.49 ± 0.13 mol/mol of NOS), by on-line gel-filtration/inductively coupled plasma mass spectrometry. Cobalt, manganese, molybdenum, nickel, and vanadium were all undetectable. Baculovirus-expressed nNOS also bound up to 2.00 ± 0.58 mol of copper/mol of NOS. Diethylenetriaminepentaacetic acid (DTPA) reduced the bound zinc to 0.28 ± 0.07 and the copper to 0.97 ± 0.24 mol/mol of NOS. Desalting of samples into thiol-free buffer did not affect the zinc content but completely eliminated the bound copper (≤0.02 mol/mol of NOS). Most (≥75%) of the bound zinc was released from baculovirus-expressed rat nNOS byp-chloromercuriphenylsulfonic acid (PMPS). PMPS-treated nNOS was strongly (90 ± 5%) inactivated. To isolate functional effects of zinc release from other effects of PMPS, PMPS-substituted thiols were unblocked by excess reduced thiol in the presence of DTPA, which hindered reincorporation of zinc. The resulting enzyme contained 0.12 ± 0.05 mol of zinc but had a specific activity of 426 ± 46 nmol of citrulline.mg−1.min−1, corresponding to 93 ± 10% of non-PMPS-treated controls. PMPS also caused dissociation of nNOS dimers under native conditions, an effect that was blocked by the pteridine cofactor tetrahydrobiopterin (H4biopterin). H4biopterin did not affect zinc release. Even in the presence of H4biopterin, PMPS prevented conversion of NOS dimers to an SDS-resistant form. We conclude that zinc binding is a prerequisite for formation of SDS-resistant NOS dimers but is not essential for catalysis.

Interference of Carboxy-PTIO with Nitric Oxide- and Peroxynitrite-Mediated Reactions

Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1, Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 = 36 +/- 5 microM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC50 of 0.12 +/- 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC50 of 0.11 +/- 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger.

Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris.

Human endothelial nitric-oxide synthase (eNOS) was expressed in the methylotrophic yeast Pichia pastoris, making use of the highly inducible alcohol oxidase promoter. The recombinant protein constituted approximately 3% of total protein and was largely soluble (>75%). About 1 mg of purified eNOS was obtained from 100-ml yeast cell cultures by affinity chromatography of crude cell supernatants. The purified enzyme had aV max of 192 ± 18 nmol ofl-citrulline × mg−1 × min−1, had a K m forl-arginine of 3.9 ± 0.2 μm, and showed an absolute requirement for tetrahydrobiopterin (H4biopterin). NADPH oxidase activity was 136 ± 9 and 342 ± 24 nmol × mg−1 × min−1 in the absence and presence of 0.1 mm l-arginine, respectively, and not affected by H4biopterin. The protein contained 0.56 ± 0.06 equivalents of FAD and 0.79 ± 0.08 equivalents of FMN. On-line gel filtration/inductively coupled plasma mass spectrometry analysis confirmed that both iron (0.80 ± 0.09 mol/subunit) and zinc (0.43 ± 0.03 mol/subunit) were bound to the enzyme. Graphite furnace-atomic absorption spectroscopy yielded a value for bound iron of 0.84 ± 0.04 mol/subunit. The absorbance of the enzyme at 398 nm implied a heme content of 0.85 ± 0.09 mol/subunit, and the high pressure liquid chromatography heme assay gave an estimate of 0.71 ± 0.02 mol heme/subunit. Gel permeation chromatography yielded one single peak with a Stokes radius of 6.62 ± 0.7 nm, indicating that the native protein is dimeric. Upon low temperature gel electrophoresis the untreated protein appeared mainly as a monomer (88 ± 3%), but pretreatment with H4biopterin andl-arginine led to a pronounced shift toward dimers (77 ± 4%). Thus, in contrast to bovine eNOS (List, B. M., Klösch, B., Völker, C., Gorren, A. C. F., Sessa, W. C., Werner, E. R., Kukovetz, W. R., Schmidt, K., and Mayer, B. (1997) Biochem. J. 323, 159–165; Rodriguez-Crespo, I., Gerber, N. C., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 11462–11467), the human eNOS appears to be markedly stabilized by H4biopterin.

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST‐tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC50 for nNOS was 18±6 μM GST‐PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K M for l‐arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.

Activation of Neuronal Nitric-oxide Synthase by the 5-Methyl Analog of Tetrahydrobiopterin FUNCTIONAL EVIDENCE AGAINST REDUCTIVE OXYGEN ACTIVATION BY THE PTERIN COFACTOR

Tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-l-biopterin (H4biopterin)) is an essential cofactor of nitric-oxide synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme group in the oxygenase domain and results in a pronounced stabilization of the active homodimeric structure of the protein. However, these allosteric effects are also produced by the potent pterin antagonist of NOS, 4-amino-H4biopterin, suggesting that the natural cofactor has an additional, as yet unknown catalytic function. Here we show that the 5-methyl analog of H4biopterin, which does not react with O2, is a functionally active pterin cofactor of neuronal NOS. Activation of the H4biopterin-free enzyme occurred in a biphasic manner with half-maximally effective concentrations of approximately 0.2 μm and 10 mm 5-methyl-H4biopterin. Thus, the affinity of the 5-methyl compound was 3 orders of magnitude lower than that of the natural cofactor, allowing the direct demonstration of the functional anticooperativity of the two pterin binding sites of dimeric NOS. In contrast to H4biopterin, which inactivates nitric oxide (NO) through nonenzymatic superoxide formation, up to 1 mmof the 5-methyl derivative did not consume O2 and had no effect on NO steady-state concentrations measured electrochemically with a Clark-type NO electrode. Therefore, reconstitution with 5-methyl-H4biopterin allowed, for the first time, the detection of enzymatic NO formation in the absence of superoxide or NO scavengers. These results unequivocally identify free NO as a NOS product and indicate that reductive O2 activation by the pterin cofactor is not essential to NO biosynthesis.

Enzymology of Nitric Oxide Biosynthesis

In mammals under normal physiological conditions, nitric oxide originates from the reaction shown in Fig. 1 (Palmer et al., 1988). The nitrogen atom comes from the guanidino group of L-arginine (Palmer et al., 1988; Sakuma et al., 1988) and the oxygen atom from molecular oxygen (Kwon et al., 1990; Leone et al., 1991). (NOHLA) has been identified as an intermediate (Stuehr et al., 1991 b), so that the reaction can be divided into two stages as shown.