Revisiting the Val/Ile Mutation in Mammalian and Bacterial Nitric Oxide Synthases: A Spectroscopic and Kinetic Study

Biochemistry. 2017 Feb 7;56(5):748-756. doi: 10.1021/acs.biochem.6b01018. Epub 2017 Jan 20.

Abstract

Nitric oxide is produced in mammals by the nitric oxide synthase (NOS) isoforms at a catalytic site comprising a heme associated with a biopterin cofactor. Through genome sequencing, proteins that are highly homologous to the oxygenase domain of NOSs have been identified, in particular in bacteria. The active site is highly conserved except for a valine residue in the distal pocket that is replaced with an isoleucine in bacteria. This switch was previously reported to influence the kinetics of the reaction. We have used the V346I mutant of the mouse inducible NOS (iNOS) as well as the I224V mutant of the NOS from Bacillus subtilis (bsNOS) to study their spectroscopic signatures in solution and look for potential structural differences compared to their respective wild types. Both mutants seem destabilized in the absence of substrate and cofactor. When both substrate and cofactor are present, small differences can be detected with Nω-hydroxy-l-arginine compared to arginine, which is likely due to the differences in the hydrogen bonding network of the distal pocket. Stopped-flow experiments evidence significant changes in the kinetics of the reaction due to the mutation as was already known. We found these effects particularly marked for iNOS. On the basis of these results, we performed rapid freeze-quench experiments to trap the biopterin radical and found the same results that we had obtained for the wild types. Despite differences in kinetics, a radical could be trapped in both steps for the iNOS mutant but only for the first step in the mutant of bsNOS. This strengthens the hypothesis that mammalian and bacterial NOSs may have a different mechanism during the second catalytic step.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Arginine / analogs & derivatives
  • Arginine / chemistry
  • Arginine / metabolism
  • Bacillus subtilis / enzymology
  • Bacillus subtilis / genetics
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biocatalysis
  • Biopterin / chemistry
  • Biopterin / metabolism
  • Catalytic Domain
  • Coenzymes / chemistry
  • Coenzymes / metabolism
  • Conserved Sequence
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Hydrogen Bonding
  • Isoleucine / chemistry*
  • Isoleucine / metabolism
  • Kinetics
  • Mice
  • Mutation*
  • Nitric Oxide / chemistry
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase / chemistry*
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type II / chemistry*
  • Nitric Oxide Synthase Type II / genetics
  • Nitric Oxide Synthase Type II / metabolism
  • Oxidation-Reduction
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Species Specificity
  • Valine / chemistry*
  • Valine / metabolism

Substances

  • Bacterial Proteins
  • Coenzymes
  • Recombinant Proteins
  • Isoleucine
  • Biopterin
  • Nitric Oxide
  • N(omega)-hydroxyarginine
  • Arginine
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, mouse
  • Valine