1 research outputs found
Regulatory Role of Glu546 in Flavin Mononucleotide î—¸ Heme Electron Transfer in Human Inducible Nitric Oxide Synthase
Nitric oxide (NO) production by mammalian
NO synthase (NOS) is believed to be regulated by the docking of the
flavin mononucleotide (FMN) domain in one subunit of the dimer onto
the heme domain of the adjacent subunit. Glu546, a conserved charged
surface residue of the FMN domain in human inducible NOS (iNOS), is
proposed to participate in the interdomain FMN/heme interactions [Sempombe
et al. <i>Inorg. Chem.</i> <b>2011</b>, <i>50</i>, 6869–6861]. In the present work, we further investigated
the role of the E546 residue in the FMN–heme interdomain electron
transfer (IET), a catalytically essential step in the NOS enzymes.
Laser flash photolysis was employed to directly measure the FMN–heme
IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN)
construct. The temperature dependence of the IET kinetics was also
measured over the temperature range of 283–304 K to determine
changes in the IET activation parameters. The E546N mutation was found
to retard the IET by significantly raising the activation entropic
barrier. Moreover, pulsed electron paramagnetic resonance data showed
that the geometry of the docked FMN/heme complex in the mutant is
basically the same as in the wild type construct, whereas the probability
of formation of such a complex is about twice lower. These results
indicate that the retarded IET in the E546N mutant is not caused by
an altered conformation of the docked FMN/heme complex, but by a lower
population of the IET-active conformation. In addition, the negative
activation entropy of the mutant is still substantially lower than
that of the holoenzyme. This supports a mechanism by which the FMN
domain can modify the IET through altering probability of the docked
state formation