Molecular mechanisms of unprecedented functions of exchangeable/labile heme and heme proteins including transcription, DNA binding, protein kinase activity, K ⁺ channel functions, cis–trans isomerization, N–N bond formation, and other functions are described.

PAS domains, which have been identified in over 1100 proteins from all three kingdoms of life, convert various input stimuli into signals that propagate to downstream components by modifying protein-protein interactions. One such protein is the Escherichia coli redox sensor, Ec DOS, a phosphodiesterase that degrades cyclic adenosine monophosphate in a redox-dependent manner. Here we report the crystal structures of the heme PAS domain of Ec DOS in both inactive Fe³⁺ and active Fe²⁺ forms at 1.32 and 1.9 Å resolution, respectively. The protein folds into a characteristic PAS domain structure and forms a homodimer. In the Fe³⁺ form, the heme iron is ligated to a His-77 side chain and a water molecule. Heme iron reduction is accompanied by heme-ligand switching from the water molecule to a side chain of Met-95 from the FG loop. Concomitantly, the flexible FG loop is significantly rigidified, along with a change in the hydrogen bonding pattern and rotation of subunits relative to each other. The present data led us to propose a novel redox-regulated molecular switch in which local heme-ligand switching may trigger a global “scissor-type” subunit movement that facilitates catalytic control.

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca²⁺ concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca²⁺, whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40–50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca²⁺ dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Δ40 and Δ42) retained maximal NO synthesis activity at lower concentrations of free Ca²⁺ than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca²⁺/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca²⁺concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca²⁺/CaM. However, reduction of heme in the Δ40 mutant occurred spontaneously on addition of NADPH in the absence of Ca²⁺/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca²⁺/CaM and by destabilizing CaM binding at low Ca²⁺ concentrations, consistent with its role as an autoinhibitory domain.

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, andSIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. EcDOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its α-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min⁻¹ with cAMP, which was optimal at pH 8.5 in the presence of Mg²⁺ and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.