Human soluble guanylate cyclase: functional expression, purification and structural characterization.

Soluble guanylate cyclase is an enzyme that catalyzes formation of cGMP from GTP and is a member of the nucleotide cyclase family of enzymes. sGC is a receptor for endogenous and exogenous nitric oxide and is activated several-fold upon its binding, constituting a core enzyme in the nitric oxide signal transduction pathway. cGMP generated by sGC is an important second messenger that regulates activity of several enzymes triggering such important physiologic reactions as vasodilation, smooth muscle relaxation and platelet aggregation. We report here the functional expression of the human isoform of soluble guanylate cyclase in HighFive insect cells using a baculovirus expression system. Highly active recombinant protein was obtained without heme reconstitution or supplementation of the cell growth medium and the level of protein expression was found to be heavily affected by the composition of the growth medium. We have successfully purified highly active sGC (sp act up to 940 nmol/min/mg) from adherent cultures using a three-column, 1-day procedure. The UV-Vis spectrum of the isolated protein shows a Soret band at 431 nm, consistent with a histidine-ligated, 5-coordinate heme as previously reported. Far UV CD spectroscopy, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dye bis-ANS, size-exclusion chromatography, and small angle X-ray scattering (SAXS) were used to characterize the structural properties of the purified sGC. We used two hierarchical neural network methods to predict the secondary structure of sGC and found it to be consistent with the observed CD spectrum of sGC.

Dietary titanium and infant growth.

Dietary titanium as TiO2+ improved animal growth during infancy while inhibiting the metabolism of intestinal bacteria. TiO2+ was also found capable of inhibiting human cytomegalovirus in tissue culture. These and other findings indicate TiO2+ improves infant growth by acting as an antibacterial and antiviral agent. The behavior of TiO2+ stands in contrast to that of TiO2, which is inert.

Characterization of human liver inducible nitric oxide synthase expressed in Escherichia coli.

We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into an Escherichia coli expression vector and have expressed and purified the enzyme. The protein has been expressed with and without a polyhistidine tail. In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers. Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence of L-arginine (L-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein. The enzyme purified in the presence of both L-Arg and H4B is highly active, with a Vmax of approximately 800 nmol NO min(-1) mg(-1) and a Km for L-Arg of 22 microM. The cytochrome c reductase activity is 38,000 nmol x min(-1) mg(-1). Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+. These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+ concentration. The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages.

Active site topologies and cofactor-mediated conformational changes of nitric-oxide synthases.

The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes. The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms. The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D. H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D. Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein. The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene. L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction. The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites.

Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation.

Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH4). In the presence of BH4, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH4-free protein is decreased by the addition of BH4 without a corresponding increase in the high-spin component. Addition of BH4 decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH4 directly modulates the heme environment. BH4-free eNOS is completely inactive, but catalytic activity is recovered when BH4 (EC50 approximately 200 nM) is added. The spectroscopically determined binding constants for L-arginine are approximately 1.9 microM in the presence and approximately 4.0 microM in the absence of BH4. The BH4-supplemented enzyme has an activity of 90-120 nmol of citrulline.min-1.mg-1 and Km values of 3 and 14 microM for L-arginine and N-hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH4-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH4-reconstituted protein. Addition of BH4, increases the percent of the dimer by only approximately 5%. The results establish that BH4 influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.

Peroxynitrite reduction of calmodulin stimulation of neuronal nitric oxide synthase.

The Ca(2+)-dependent binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) stimulates the catalytic oxidation of L-arginine to nitric oxide. The CaM-dependent increase in catalytic activity is associated with an increase in the flow of electrons from the flavoprotein to the heme domain. In the presence of suboptimal arginine concentrations, uncoupled turnover of nNOS produces both nitric oxide and superoxide, reactive species which combine to form peroxynitrite. We demonstrate here that peroxynitrite and other oxidants produced by nNOS oxidize the methionine residues of CaM and show that the ability of CaM to stimulate nNOS is impaired by this oxidative modification. Of the nine Met residues, those at the C-terminus (Met-144, -145, -124, -109) are most sensitive to oxidation. Correlation of the Met oxidation pattern with ability to stimulate nNOS suggests that oxidation of Met-36 is particularly important for the stimulation of nNOS. Incubation of nNOS with suboptimal concentrations of arginine results in sulfoxidation of the CaM methionine residues. Although nitration of the tyrosine residues in CaM could also occur, this does not occur to a significant extent in the present system. The results suggest that peroxynitrite may exert a feedback effect on its own formation by oxidizing CaM and thereby decreasing its ability to stimulate the turnover of nNOS.

Neuronal nitric oxide synthase. Expression in Escherichia coli, irreversible inhibition by phenyldiazene, and active site topology.

A gene coding for rat neuronal nitric oxide synthase (nNOS) has been cloned into pCWori and the vector has been expressed in Escherichia coli. The expressed enzyme has been purified with a final yield of purified protein of approximately 1 mg/g of wet cells. The recombinant protein reconstituted with calmodulin and Ca2+ exhibits spectroscopic and catalytic properties identical to those reported in the literature for nNOS. Reaction of recombinant nNOS with phenyldiazene produces a phenyl-iron (Fe.Ph) complex with a maximum at 470 nm. Formation of this complex is paralleled by inactivation of the enzyme and is inhibited by arginine, the natural substrate of the enzyme. Phenyl-iron complex formation does not alter the rate of electron transfer from the flavin domain to cytochrome c. Addition of ferricyanide triggers migration of the phenyl residue from the iron to the porphyrin nitrogens. The N-phenylprotoporphyrin isomers with the phenyl on the nitrogens of pyrrole rings B, A, C, and D are formed in, respectively, approximately a 14:20:21:45 ratio. The regioisomer pattern indicates that the active site of NOS is open to some extent above all four pyrrole rings but more so above pyrrole ring D. Arylhydrazines are thus not only a new class of inhibitors of nNOS but provide useful information on the active site topology of the enzyme.

Electrostatic control of the substrate access channel in cytochrome P-450cam.

Camphor binding to ferric cytochrome P-450cam is a two-step process. The first step corresponds to the diffusion of camphor into the heme pocket, and the second one corresponds to an observable spin transition of the heme iron. In this paper, electrostatic interactions that may control the opening of the structure to allow substrate access to the buried and not solvent-exposed active site were examined. The electrostatic interactions occurring at the protein surface were weakened by increasing the ionic strength of the medium with sodium salts and strengthened by decreasing the dielectric constant of the medium with ethylene glycol as a cosolvent. The results obtained with the wild-type protein were compared to those obtained with the site-directed mutant of cytochrome P-450cam in which the Arg 186-Asp 251 and Lys 178-Asp 251 salt bridges, located at the entrance of the proposed access channel, were suppressed by replacing Asp 251 with an asparagine residue. Over a range of sodium chloride concentrations from 0 to 400 mM, camphor binding is favored, as seen in the variation in the first step dissociation equilibrium constant, K1d, which decreases from 49.5 to 24 microM, respectively. Addition of ethylene glycol favors the dissociation of the substrate-bound complex. The addition of sodium to the ethylene glycol-containing samples reverses the effect of the cosolvent. Removal of the Arg 186-Asp 251 and Lys 178-Asp 251 salt bridges results in an alteration in camphor binding in which K1d is equal to 34 microM without sodium.(ABSTRACT TRUNCATED AT 250 WORDS)

A role for Asp-251 in cytochrome P-450cam oxygen activation.

We have mutated Asp-251, Thr-252, and Lys-178 in cytochrome P-450cam and studied their effect on steady-state P-450cam catalysis. The mutation of Asp-251 to Asn, which dramatically slows the reaction rate, affects a pH-dependent step in the reaction cycle. By examining the individual steps in the reaction cycle, we have determined that the effect of the D251N mutation occurs after dioxygen binding. Furthermore, our results suggest that the rate-limiting step of the D251N reaction cycle is the O-O bond scission event and that this residue also plays a crucial role in O-O bond scission in wild-type P-450cam. Based on homology with other P-450 enzymes and previous mutagenesis investigations, this role may be common to other P-450 systems, and we suggest a mechanism that is consistent with the effects of these mutations on enzyme activity.

Identification of 2Fe-2S cysteine ligands in putidaredoxin.

The iron-sulfur center of putidaredoxin is coordinated by four cysteine sulfhydrals. In order to determine which of the six cysteine residues in the protein coordinate the Fe-S center, we have individually mutated cysteine residues 73, 85 and 86 into serines. Of these mutant proteins, only C85S and C73S express holo-protein as evidence by SDS-PAGE and EPR spectroscopy. This leads us to the conclusion that residues 39,45,48, and 86 are the cysteines that coordinate the iron-sulfur center in putidaredoxin.