ESEEM and ENDOR studies of the Rieske iron-sulphur protein in bovine heart mitochondrial membranes.

Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) were applied to the respiratory-chain iron-sulphur clusters in natural bovine heart mitochondrial membranes. By using specific reduction, signals were observed from the Complex III Rieske [2Fe-2S] cluster. In ENDOR, 1H hyperfine couplings in the range 0.5-7 MHz were observed. In ESEEM, modulations were obtained which were assigned to two 14N nuclei of directly-coupled imidazole ligands. The ESEEM spectra are similar to previous observations on purified iron-sulphur proteins of this type, in which the iron-sulphur cluster is coordinated by two cysteine and two histidine ligands. They confirm that the coordination of the cluster in the purified proteins, with two cysteinyl sulphur and two histidine nitrogens, is unchanged from its natural mitochondrial membrane environment. In order to investigate the possible interaction of the membrane-bound Rieske protein with quinones, measurements were conducted on membranes preincubated with 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), and in the pH range 6-7.5. No significant changes were detected, either in the proton hyperfine couplings as detected by ENDOR, or in the nitrogen couplings to the histidines as detected by ESEEM.

ESEEM studies of the iron-sulphur clusters of succinate dehydrogenase in Arum maculatum spadix mitochondrial membranes.

We have performed ESEEM spectroscopy in order to obtain structural information about the environment of the [2Fe-2S] cluster and the [3Fe-4S] cluster of succinate dehydrogenase (Centres 1 and 3, respectively) in intact Arum maculatum mitochondrial membranes. Both iron-sulphur clusters showed modulations indicative of 14N in the three-pulse echo decay envelopes. We have estimated the hyperfine couplings for the reduced [2Fe-2S] cluster (A approximately 1.1 MHz) and the oxidised [3Fe-4S] cluster (A approximately 0.8 MHz). Our results are compared with ESEEM data obtained for purified [2Fe-2S] cluster-containing proteins, namely Spirulina platensis ferredoxin, a protein for which the three-dimensional structure is known, and Escherichia coli fumarate reductase. The hyperfine and quadrupolar coupling parameters determined are consistent with a weak interaction of Centre 1 and Centre 3 with peptide 14N, rather than 14N of imidazole rings.

L-arginine depletion by arginase reduces nitric oxide production in endotoxic shock: an electron paramagnetic resonance study.

Nitric oxide (NO) synthesis was measured in the liver, lung, spleen and kidney of lipopolysaccharide-treated male rats using the nitric oxide spin trap, iron (II)-diethyldithiocarbamate (FeDETC2). Nitric oxide formation in vivo was determined by the increase in intensity of the characteristic triplet hyperfine EPR spectrum of [NO-FeDETC2]. Intravenous bovine liver arginase, at a dose which completely depleted circulating arginine, significantly reduced the formation of nitric oxide in these tissues. The general decrease in NO levels was confirmed by the decrease in plasma nitrite levels. These results directly demonstrate that NO formation in endotoxic shock depends on extracellular arginine; depletion of plasma arginine may be a useful therapeutic strategy.

Blue and yellow laccases of ligninolytic fungi.

Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.

2, 4-diamino-6- hydroxy pyrimidine inhibits NSAIDs induced nitrosyl-complex EPR signals and ulcer in rat jejunum.

BACKGROUND: It has been suggested that one aspect of non-steroidal anti-inflammatory drugs induced intestinal damage is due to either uncoupling of mitochondrial oxidative phosphorylation or inhibition of electron transport. We investigated the latter possibility using electron paramagnetic resonance spectroscopy. RESULTS: Electron paramagnetic studies of NSAIDS on sub-mitochondrial particles revealed that indomethacin, but not with nabumetone, bound to a site near to Complex I and ubiquinone to generate a radical species. Normal rats exhibited prominent [3Fe-4S]ox signals (g approximately 2.01) at 20 K. One hour after indomethacin there was a prominent, intense and broad absorption pattern at (g approximately 2.07) suggesting, appearance of radical species overlapping [3Fe-4S]ox and was unaffected by pretreatment with 2,4 diamino -6-hydroxy pyrimidine. At 24 hrs, when macroscopic ulcers were seen, there was a new signal due to a nitric oxide radical (NO*). In contrast, nabumetone and 2,4 diamino-6-hydroxy pyrimidine pre-treated animals receiving indomethacin exhibited electron paramagnetic resonance spectra identical to those of controls at 24 hrs and neither was associated with small intestinal ulcers. Indomethacin and 2,4 diamino hydroxy pyrimidine pre-treated rats, but not nabumetone, had increased intestinal permeability. CONCLUSION: The results suggest that the in vivo effects of indomethacin modulate the mitochondrial respiratory chain directly at 1 h and 24 h through formation of nitric oxide. NO* appears to play an important role in the late pathogenic stages of NSAID enteropathy and may be the site for targeted treatment to reduce their toxicity.

A helical region on human lactoferrin. Its role in antibacterial pathogenesis.

Human lactoferrin contains a 47 amino acid peptide, named lactoferricin H, which is thought to be responsible for its antimicrobial activity. Lactoferricin includes a loop region, which resides on the outer surface of the N-lobe of lactoferrin, adopting an alpha helix with a hydrophobic tail. Peptides have been synthesised corresponding to the highly charged alpha helix (HLP 2) and hydrophobic tail region (HLP 5). HLP 2 has potent antibacterial activity whereas HLP 5 had no activity. To investigate the relationship between structure and function of HLP 2, HLP 6 was synthesised with a proline replacing methionine. This substitution was predicted to disrupt the helical region of the peptide and the orientation of the positively charged residues. Antibacterial activity was significantly reduced when tested against Escherichia coli serotype 0111, NCTC 8007. The mode of action of HLP 2 against the bacterial membrane was investigated by flow cytometric analysis, using Escherichia coli, NCTC 8007. Membrane potential and integrity were monitored using the fluorescent probes, bis 1,3-(dibutylbarbituric acid) trimethine oxonol and propidium iodide respectively. HLP 2 caused complete loss of membrane potential and integrity, with irreversible damage to the cell as shown by rapid loss of viability. We conclude that HLP 2 causes membrane disruption and that helicity is an important factor for antibacterial activity.

Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide.

Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)2Fe(NO)2. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron-sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.

Coordination of the Rieske-type [2Fe-2S] cluster of the terminal iron-sulfur protein of Pseudomonas putida benzene 1,2-dioxygenase, studied by one- and two-dimensional electron spin-echo envelope modulation spectroscopy.

One- and two-dimensional (1D and 2D) electron spin-echo envelope modulation (ESEEM) spectroscopy has been used to investigate the ligand environment of the [2Fe-2S] cluster from the terminal dioxygenase (ISPBED) of the Pseudomonas putida benzene dioxygenase complex. The modulation frequencies observed in the 0.5-8.5 MHz region of the Fourier transforms of 1D and 2D ESEEM spectra measured across the electron paramagnetic resonance (EPR) absorbance envelope (from gz through to gx) are consistent with their assignment to two 14N nuclei. Using hyperfine sublevel correlation spectroscopy (HYSCORE), two sets of correlated double quantum transitions sharing the same hyperfine coupling were observed and were identified as being due to the same two 14N nuclei. On the basis of the isotropic hyperfine and quadrupolar couplings estimated for these 14N nuclei [N(1), Aiso = 3.6 MHz and e2qQ = 2.2-2.8 MHz; N(2), Aiso = 4.8 MHz and e2qQ = 2.2-2.4 MHz], the ESEEM pattern of ISPBED is assigned to two histidine nitrogens which are directly coordinated to the reduced iron-sulfur cluster. Bonding parameters of the two [14N]histidine ligands were calculated from these hyperfine couplings. The primary covalent contributions to the hyperfine interaction arise from 14N-to-Fe2+ sigma bonds. For N(1), our analysis of the percentage of unpaired 2s and 2p electrons gave f2s approximately 1.3% and f2p approximately 0.2%, while values of f2s approximately 1.7% and f2p approximately 1.4% were estimated for N(2). Comparison of these values with those determined from electron nuclear double resonance (ENDOR) data of the Rieske-type [2Fe-2S] center of Pseudomonas cepacia phthalate dioxygenase [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871] indicates an apparent reduction in unpaired electron spin density residing on the two 14N ligands of ISPBED. Analysis of slices of the HYSCORE spectrum has provided evidence for another 14N nucleus (A approximately 1.1 MHz, e2qQ = 3.3 MHz), which we have attributed to a weakly coupled peptide nitrogen, similar to those observed for ferredoxin-type [2Fe-2S] clusters. This type of weak interaction has not been previously described by the detailed ENDOR and ESEEM studies of Rieske-type centers. The resolution of the spectra demonstrates the effectiveness of 2D ESEEM for the disentanglement of multiple hyperfine interactions to metalloprotein centers.

Coordination of the [2Fe-2S] cluster in wild type and molecular variants of Clostridium pasteurianum ferredoxin, investigated by ESEEM spectroscopy.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56, and 60. This pattern is unique, and a combination of site-directed mutagenesis and spectroscopy is therefore being implemented to identify the ligands of the [2Fe-2S] cluster. The possible involvement of ligands other than cysteine in some molecular variants of this ferredoxin has been considered, histidines being likely candidates. Therefore, the three histidine residues in positions 6, 7, and 90 of the amino acid sequence have been individually and collectively replaced by alanine or valine. The mutated ferredoxins have been purified and were all found to contain [2Fe-2S] clusters of which the UV-visible absorption spectra were identical to that of the wild-type protein. The H6A/H7A/ H90A triply mutated ferredoxin was further characterized by EPR and by ESEEM spectroscopy and was found to differ only marginally from the wild-type protein. The ESEEM spectra of wild-type ferredoxin displayed weak 14N hyperfine interactions at the three principal g-factors of the [2Fe-2S] center. The estimated 14N coupling constants (Aiso = 0.6 MHz; e2qQ approximately 3.3 MHz) indicate that the ESEEM effect is most likely due to 14N from the polypeptide backbone. 2H2O ESEEM spectra showed that the [2Fe-2S] cluster is accessible for exchange with solvent deuterons. ESEEM spectra of the previously characterized C24A and C14A/C24A variants have been recorded and were also found to be very similar to those of the wild-type protein. There was no evidence for coordination of the [2Fe-2S] cluster by [14N]histidine or other 14N nuclei, in either wild-type or mutant forms of the ferredoxin. By these criteria, the environment of the [2Fe-2S] center is not distinguishable from those in plant-type ferredoxins. Non-cysteinyl coordination most probably occurs only in the C14A/C24A variant, which contains no more than three cysteine residues. The data shown here indicate that the fourth ligand of the [2Fe-2S] cluster is neither a histidine residue nor another nitrogenous ligand. The possibility of oxygenic coordination for this molecular variant is discussed.

Oxidative metabolism of inorganic sulfur compounds by bacteria.

The history of the elucidation of the microbiology and biochemistry of the oxidation of inorganic sulfur compounds in chemolithotrophic bacteria is briefly reviewed, and the contribution of Martinus Beijerinck to the study of sulfur-oxidizing bacteria highlighted. Recent developments in the biochemistry, enzymology and molecular biology of sulfur oxidation in obligately and facultatively lithotrophic bacteria are summarized, and the existence of at least two major pathways of thiosulfate (sulfur and sulfide) oxidation confirmed. These are identified as the 'Paracoccus sulfur oxidation' (or PSO) pathway and the 'S4intermediate' (or S4I) pathway respectively. The former occurs in organisms such as Paracoccus (Thiobacillus) versutus and P. denitrificans, and possibly in Thiobacillus novellus and Xanthobacter spp. The latter pathway is characteristic of the obligate chemolithotrophs (e.g. Thiobacillus tepidarius, T. neapolitanus, T. ferrooxidans, T. thiooxidans) and facultative species such as T. acidophilus and T. aquaesulis, all of which can produce or oxidize tetrathionate when grown on thiosulfate. The central problem, as yet incompletely resolved in all cases, is the enzymology of the conversion of sulfane-sulfur (as in the outer [S-] atom of thiosulfate [-S-SO3-]), or sulfur itself, to sulfate, and whether sulfite is involved as a free intermediate in this process in all, or only some, cases. The study of inorganic sulfur compound oxidation for energetic purposes in bacteria (i.e. chemolithotrophy and sulfur photolithotrophy) poses challenges for comparative biochemistry. It also provides evidence of convergent evolution among diverse bacterial groups to achieve the end of energy-yielding sulfur compound oxidation (to drive autotrophic growth on carbon dioxide) but using a variety of enzymological systems, which share some common features. Some new data are presented on the oxidation of 35S-thiosulfate, and on the effect of other anions (selenate, molybdate, tungstate, chromate, vanadate) on sulfur compound oxidation, including observations which relate to the roles of polythionates and elemental sulfur as intermediates.

Probing magnetic properties of the reduced [2Fe-2S] cluster of the ferredoxin from Arthrospira platensis by 1H ENDOR spectroscopy.

The 1H electron nuclear double resonance (ENDOR) spectra in frozen solutions of the reduced [2Fe-2S] cluster in ferredoxin from Arthrospira (Spirulina) platensis have been measured at low temperatures (5-20 K) and simulated using orientational selection methods. The analysis confirmed the existence of a single paramagnetic species with iron valence states II and III connected uniquely to the cluster irons. The experimental ENDOR spectra were fitted to a model including the spin distribution on the centre, the orientation of the g-matrix, and the isotropic and anisotropic hyperfine couplings of the nearest protons in the crystallographically determined structure. In order to partially simulate ENDOR line shapes, a statistical distribution of the corresponding torsion angles between the Fe(III) centre and one of the beta-CH2 protons was introduced. From the analysis, four of the larger hyperfine couplings found were assigned to the cysteine beta-protons near the Fe(III) ion of the cluster, with isotropic hyperfine couplings ranging from 1.6 to 4.1 MHz. The spin distribution on the two iron ions was estimated to be +1.85 for the Fe(III) ion and -0.9 for the Fe(II) ion. The Fe(III) ion was identified as being coordinated to the cysteine ligands Cys49 and Cys79, confirming previous NMR results. The direction of the g-tensor with respect to the cluster was deduced. The g1-g2 plane is parallel to the planes through each iron and its adjacent cysteine sulfurs; the g2-g3 plane is nearly perpendicular to the latter planes and deviates by 25 degrees from the FeSSFe plane. The g1 direction is dominated by the bonding geometry of Fe(II) and does not align with the Fe(II)-Fe(III) vector.

EPR spectroscopic characterization of the iron-sulphur proteins and cytochrome P-450 in mitochondria from the insect Spodoptera littoralis (cotton leafworm).

EPR spectroscopy was used to investigate the cytochrome P-450-dependent steroid hydroxylase ecdysone 20-mono-oxygenase of the cotton leafworm (Spodoptera littoralis) and the redox centres associated with membranes from the fat-body mitochondrial fraction. Intense features at g = 2.42, 2.25 and 1.92 from oxidized mitochondrial membranes have been assigned to the low-spin haem form of ferricytochrome P-450, probably of ecdysone 20-mono-oxygenase. High-spin cytochrome P-450 (substrate-bound) was tentatively assigned to a signal at g = 8.0, which was detectable from membranes as prepared. An EPR signal characteristic of a [2Fe-2S] cluster detected from the soluble mitochondrial matrix fraction has been shown to be distinct from the signals associated with mitochondrial NADH dehydrogenase and succinate dehydrogenase, and has therefore been attributed to a ferredoxin. We conclude that the S. littoralis fat-body mitochondrial electron-transport system involved in steroid 20-hydroxylation comprises both ferredoxin and cytochrome P-450 components, and thus resembles the enzyme systems of adrenocortical mitochondria. EPR signals characteristic of the respiratory chain were also observed from fat-body mitochondria and assigned to the iron-sulphur clusters associated with Complex I (Centres N1, N2), Complex II (Centres S1, S3), Complex III (the Rieske centre), and the copper centre of Complex IV, demonstrating similarities to mammalian mitochondria. The reduced membrane fraction also yielded a major resonance at g = 2.09 and 1.88 characteristic of the [4Fe-4S] cluster of electron-transferring flavoprotein: ubiquinone oxidoreductase. As the fat-body is the major metabolic organ of insects, this protein is presumably required for the beta-oxidation of fatty acids in mitochondria. High-spin haem signals in the low-field region of spectra also demonstrated that the mitochondrial fraction contains relatively high concentrations of catalase.

Detection of nitrosyl complexes in human substantia nigra, in relation to Parkinson's disease.

Idiopathic Parkinson's disease (PD) involves a documentable decline in the activity of mitochondrial complex I in substantia nigra (1-3). We have EPR spectroscopy to investigate complex I in human substantia nigra and globus pallidus. EPR signals characteristic of the iron-sulfur centers of complexes I and II were observed with globus pallidus, with no significant difference between control and PD. These complex 1 signals could not be clearly observed in substantia nigra. Instead, nitric oxide (NO.) radicals in PD nigra were detected at g approximately 2.08, 1.98 due to [haem-NO] formation. Although an EPR signal indicative of haem-NO was observed with control nigra, it lacked the distinctive g approximately 1.98 trough observed with PD nigra. As PD is associated with a reactive gliosis, the difference in the haem-NO EPR signal, between control and PD nigra, may result from cytotoxic NO. generated by microglia in PD substantia nigra.

A dynamic model of the meningococcal transferrin receptor.

Iron is an essential nutrient for all organisms and consequently, the ability to bind transferrin and sequester iron from his source constitutes a distinct advantage to a blood-borne bacterial pathogen. Levels of free iron are strictly limited in human serum, largely through the action of the iron-binding protein transferrin. The acquisition of trasferrin-iron is coincident with pathogenicity among Neisseria species and a limited number of other pathogens of human and veterinary significance. In Neisseria meningitidis, transferrin binding relies on two co-expressed, outer membrane proteins distinct in aspects of both structure and function. These proteins are independently and simultaneously capable of binding human transferrin and both are required for the optimal uptake of iron from this source. It has been established that transferrin-binding proteins (designated TbpA and TbpB) form a discrete, specific complex which may be composed of a transmembrane species (composed of the TbpA dimer) associated with a single surface-exposed lipoprotein (TbpB). This more exposed protein is capable of selectively binding iron-saturated transferrin and the receptor complex has ligand-binding properties which are distinct from either of its components. Previous in vivo analyses of N. gonorrhoeae, which utilizes a closely related transferrin-iron uptake system, indicated that this receptor exists in several conformations influenced in part by the presence (or absence) of transferrin. Here we propose a dynamic model of the meningococcal transferrin receptor which is fully consistent with the current data concerning this subject. We suggest that TbpB serves as the initial binding site for iron-saturated transferrin and brings this ligand close to the associated transmembrane dimer, enabling additional binding events and orientating transferrin over the dual TbpA pores. The antagonistic association of these receptor proteins with a single ligand molecule may also induce conformational change in transferrin, thereby favouring the release of iron. As, in vivo, transferrin may have iron in one or both lobes, this dynamic molecular arrangement would enable iron uptake from either iron-binding site. In addition, the predicted molecular dimensions of the putative TbpA dimer and hTf are fully consistent with these proposals. Given the diverse data used in the formulation of this model and the consistent characteristics of transferrin binding among several significant Gram-negative pathogens, we speculate that such receptor-ligand interactions may be, at least in part, conserved between species. Consequently, this model may be applicable to bacteria other than N. meningitidis.

Comparison of the "Rieske" [2Fe-2S] center in the bc1 complex and in bacterial dioxygenases by circular dichroism spectroscopy and cyclic voltammetry.

Two different types of "Rieske" [2Fe-2S] clusters have been observed in proteins, one in the bc complexes of the respiratory chain and the other in bacterial dioxygenases. We have compared the circular dichroic (CD) spectra and redox properties of the water soluble fragment of the Rieske center of the bovine heart mitochondrial bc1 complex (ISF) and of the ferredoxin from benzene dioxygenase in Pseudomonas putida ML2 (FDBED). Spinach ferredoxin was also measured for comparison. The redox potential of both proteins could be determined in solution by cyclic voltammetry (CV) and by CD-monitored spectroelectrochemistry using a specially constructed optically transparent thin layer (OTTLE) cell. Whereas the redox potential of the ISF (+312 +/- 5 mV at pH 7.0) depended both on the pH above pH 7 and on the ionic strength, the redox potential of the FDBED (-155 +/- 5 mV at pH 7.0) was observed to be independent of pH and ionic strength. The ISF showed a marked dependence of its redox potential on temperature, while the FDBED showed no temperature dependence. The entropy of the redox reaction delta S degrees rc was calculated as -88 +/- 11 J K-1 mol-1 for the bc1 Rieske center and approximately 0 J K-1 mol-1 for the FdBED. The CD spectra of Rieske type clusters are significantly different from those of plant type [2Fe-2S] ferredoxins. A strong negative CD band is present at 20 000 cm-1 (500 nm) in all reduced Rieske clusters. The possible assignment of this band is discussed as arising from the highest energy magnetically allowed d --> d transition (dz2 --> dxz) of the FeII site. If so, this band is highly indicative of the distortion of the ligand field of the FeII site.

Inhibition of tetrahydrobiopterin synthesis reduces in vivo nitric oxide production in experimental endotoxic shock.

Nitric oxide synthesis requires the cofactor tetrahydrobiopterin. We have examined the effect on nitric oxide synthesis in experimental endotoxic shock of 2,4- diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, the first and rate limiting enzyme for tetrahydrobiopterin synthesis. Rats given lipopolysaccharide (LPS, 10 mg/kg) showed a large rise in plasma nitrate at 4 and 8 hours which was significantly reduced by DAHP (1 g/kg) given at the same time as LPS. There was a 40-50% reduction in the haem-NO signal detected in kidney by electron paramagnetic resonance spectroscopy. LPS produced hypotension at 3 hours and 6 hours and this was ameliorated at 6 hours in rats given DAHP. DAHP abolished the rise in kidney tetrahydrobiopterin levels seen 4 hours after LPS but no effect was seen on induction of inducible nitric oxide synthase (iNOS) as assessed by immunohistochemistry and reverse transcriptase PCR, consistent with the effect of DAPH being by reduction of tetrahydrobiopterin levels. The results show that inhibition of tetrahydrobiopterin synthesis is an effective strategy to reduce nitric oxide synthesis by iNOS in vivo.