Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme.

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN-heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808-3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 +/- 5 and 35 +/- 3 s(-1), respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS.

Cloning, Expression, and Purification of a Nitric Oxide Synthase-Like Protein from Bacillus cereus.

The nitric oxide synthase-like protein from Bacillus cereus (bcNOS) has been cloned, expressed, and characterized. This small hemeprotein (356 amino acids in length) has a mass of 43 kDa and forms a dimer. The recombinant protein showed similar spectral shifts to the mammalian NOS proteins and could bind the substrates L-arginine and N(G)-hydroxy-L-arginine as well as the ligand imidazole. Low levels of activity were recorded for the hydrogen peroxide-dependent oxidation of N(G)-hydroxy-L-arginine and L-arginine by bcNOS, while a reconstituted system with the rat neuronal NOS reductase domain showed no activity. The recombinant bcNOS protein adds to the complement of bacterial NOS-like proteins that are used for the investigation of the mechanism and function of NO in microorganisms.

Electron transfer in a human inducible nitric oxide synthase oxygenase/FMN construct co-expressed with the N-terminal globular domain of calmodulin.

The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a truncated calmodulin (CaM) construct that includes only its N-terminal globular domain consisting of residues 1-75, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly fourfold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca(2+) depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca(2+)-dependent reorganization of the truncated CaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of the IET.

Effect of competitiveness on forty-yard dash performance in college men and women.

The objective of this study was to determine performance differences between individual and competitive trials of the 40-yard dash. Physically active college men (n = 25) and women (n = 29) performed an individual 40-yard dash, followed by completion of the Sports Competition Trait Inventory (SCTI) before performing a paired 40-yard dash against a time-matched competitor. All sprints were performed on an indoor rubberized track using photoelectric gates to start and stop a digital timer. In addition, 3 timers used hand-held stopwatches to record the individual sprint time. There was no significant difference (p = 0.10) between men (120.3 +/- 16.6) and women (111.7 +/- 20.3) on the SCTI. There was no significant difference between individual and competitive 40-yard dash times for either men (5.21 +/- 0.24 and 5.19 +/- 0.23 seconds, respectively) or women (6.12 +/- 0.31 and 6.11 +/- 0.32 seconds, respectively). The correlation between SCTI and both individual and competitive 40-yard dashes was significant (p < 0.05) for women (r = -0.45 and -0.44, respectively) but not for men (r = -0.10 and 0.10, respectively). Electronic times (5.70 +/- 0.54 seconds) were not significantly different from 1 hand-timer (5.71 +/- 0.56 seconds) but were significantly faster than the other 2 timers (5.80 +/- 0.58 and 5.82 +/- 0.57 seconds). Averaging the 3 hand times (5.78 +/- 0.56 seconds) for comparison with the electronic timing (5.70 +/- 0.54 seconds) produced a high correlation (r = 0.96) but a significantly slower time (p < 0.05). A competitive environment does not appear to improve short sprint times in either men or women. In addition, hand timing may not always produce faster times compared to electronic timing.