Abstract
Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (Fe(III)NO). Although the NOS-like protein from Bacillus subtilis (bsNOS) generates Fe(III)NO from the reaction intermediate N-hydroxy-l-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe(II)O(2)) intermediate, Fe(III)NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe(III)NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe(II)O(2), a mixture of Fe(III)NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe(III)NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The maximal absorbance values for the Soret peak that we observed in the absence of Arg and H4B indicate that the mutants each mimicked their wild-type counterpart in that the V346I iNOSoxy had its ferric heme poised in a predominantly low spin state, whereas the I224V bsNOS had its heme poised in a predominantly high spin state
These results are consistent with wild-type iNOSoxy and bsNOS enzymes being mostly similar in these regards despite their containing either a Val or Ile at the same position
Summary
Materials—All reagents and materials were obtained from Sigma, Aldrich, Alexis, or sources described previously [19]. The Ks value of Arg was calculated using equation 1, Ks ϭ Kobs/{1 ϩ [imidazole]/Kd(imidazole)}, in which Kobs is the apparent binding constant determined for Arg. Peroxide Assay—H2O2-dependent NOHA oxidation assays were performed as described previously [20]. Enzymes were incubated at room temperature with NOHA, dithiothreitol, and different concentrations of H4B in 96-well plates. An anaerobic solution that contained the dithionite-reduced enzyme at concentrations indicated in the text, 40 mM Hepes, 0.5 or 0.2 mM NOHA, and 0.2 mM H4B was transferred into the stopped-flow instrument and rapidly mixed with air-saturated Hepes buffer at 10 °C. Anaerobic buffered solutions containing 2 M ferric enzyme, 400 M H4B, 1.2 mM dithiothreitol, and 400 M NOHA were rapidly mixed at 10 °C in the stopped-flow instrument with a buffered solution containing different concentrations of NO. The NO solutions were made by diluting a chilled saturated NO solution in chilled anaerobic buffer and assuming a NO concentration of 2 mM for a saturated solution at 10 °C
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