Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme

Abstract

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.

Figure 1

Tethered shuttle model: FMN-binding domain shuttles between the NADPH-FAD-binding domain and the heme-containing oxygenase domain. Top: putative input state; bottom: putative output state. The putative output state is envisioned as a complex between oxygenase and FMN binding domains, and its structure has not yet been elucidated. CaM binding unlocks the input state, thereby enabling the FMN domain to shuttle between the two enzyme states. In the holoenzyme the rate-limiting step in the IET is the conversion of input state to output state. The FMN domain in the iNOS holoenzyme exists predominantly in the input state, as demonstrated in this study.

Figure 2

(a) Difference and (b) absorption spectra of murine iNOS holoenzyme in the presence of 20 μM dRF obtained after approximately 1 min of steady light illumination. In panel b, dashed line, oxidized iNOS; solid line, partially reduced iNOS; note the characteristic absorption of oxidized flavin at 480 nm, as indicated by an arrow. Anaerobic solutions contained 2.4 μM iNOS, ~ 20 μM dRF and 5 mM fresh semicarbazide in pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 μM H₄B, 1 mM Ca²⁺, and 10 % glycerol). The sample was well degassed by Ar/CO (3:1) before illumination.

Figure 2

(a) Difference and (b) absorption spectra of murine iNOS holoenzyme in the presence of 20 μM dRF obtained after approximately 1 min of steady light illumination. In panel b, dashed line, oxidized iNOS; solid line, partially reduced iNOS; note the characteristic absorption of oxidized flavin at 480 nm, as indicated by an arrow. Anaerobic solutions contained 2.4 μM iNOS, ~ 20 μM dRF and 5 mM fresh semicarbazide in pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 μM H₄B, 1 mM Ca²⁺, and 10 % glycerol). The sample was well degassed by Ar/CO (3:1) before illumination.

Figure 3

Transient trace at 580 nm at (a) 0 – 0.2 s and (b) 0 – 5 s obtained for [Fe(II)−CO][FMNH^•] form of the murine iNOS holoenzyme flashed by 450 nm laser excitation. Inset of panel a is of the [Fe(II)−CO] form of the iNOSoxy construct (at 0–1 s) flashed by 450 nm laser excitation; note that the trace remains above the pre-flash baseline. Anaerobic solutions contained 7.4 μM iNOS, ~ 20 μM dRF and 5 mM fresh semicarbazide in pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 μM H₄B, 1 mM Ca²⁺ and 10 % glycerol).

Figure 3

Transient trace at 580 nm at (a) 0 – 0.2 s and (b) 0 – 5 s obtained for [Fe(II)−CO][FMNH^•] form of the murine iNOS holoenzyme flashed by 450 nm laser excitation. Inset of panel a is of the [Fe(II)−CO] form of the iNOSoxy construct (at 0–1 s) flashed by 450 nm laser excitation; note that the trace remains above the pre-flash baseline. Anaerobic solutions contained 7.4 μM iNOS, ~ 20 μM dRF and 5 mM fresh semicarbazide in pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 μM H₄B, 1 mM Ca²⁺ and 10 % glycerol).

Figure 4

Transient trace at 465 nm (and 430 nm, inset) at (a) 0 – 0.2 s and (b) 0 – 1 s obtained for the [Fe(II)−CO][FMNH^•] form of the murine iNOS holoenzyme flashed by 450 nm laser excitation. Note both traces possess a transition (i.e. a reversal in direction of absorption changes over time, panel b), as was the case for the 580 nm trace (Figure 3b). Experimental conditions were the same as Figure 3.

Figure 4

Transient trace at 465 nm (and 430 nm, inset) at (a) 0 – 0.2 s and (b) 0 – 1 s obtained for the [Fe(II)−CO][FMNH^•] form of the murine iNOS holoenzyme flashed by 450 nm laser excitation. Note both traces possess a transition (i.e. a reversal in direction of absorption changes over time, panel b), as was the case for the 580 nm trace (Figure 3b). Experimental conditions were the same as Figure 3.

Figure 5

Transient traces at 580 nm at (a) 0 – 0.2 s and (b) 0 – 2 s obtained for the [Fe(II)−CO][FMNH^•] form of a human iNOS oxyFMN construct flashed by 450 nm laser excitation. Anaerobic solutions contained 8.2 μM human iNOS oxyFMN, ~ 20 μM dRF and 5 mM fresh semicarbazide in pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 μM H₄B, 1 mM Ca²⁺ and 10 % glycerol). Note the significant difference in the time scale of the panel a and Figure 3a.

Figure 5

Transient traces at 580 nm at (a) 0 – 0.2 s and (b) 0 – 2 s obtained for the [Fe(II)−CO][FMNH^•] form of a human iNOS oxyFMN construct flashed by 450 nm laser excitation. Anaerobic solutions contained 8.2 μM human iNOS oxyFMN, ~ 20 μM dRF and 5 mM fresh semicarbazide in pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 μM H₄B, 1 mM Ca²⁺ and 10 % glycerol). Note the significant difference in the time scale of the panel a and Figure 3a.

Scheme 1

Summary of processes occurring upon CO photolysis in the partially reduced form [Fe(II)−CO][FMNH^•] of iNOS holoenzymes. Species in the dashed boxes are CO-bound forms, whereas those in the solid boxes are CO-free and participate in the FMN–heme IET (reaction 3). This IET process can be followed at 580 nm (which is due to net reduction of FMNH^•) and 465/430 nm (which is due to net oxidation of heme).