Comparing the Temperature Dependence of FMN to Heme Electron Transfer in Full Length and Truncated Inducible Nitric Oxide Synthase

Abstract

The FMN–heme intraprotein electron transfer (IET) kinetics in full length and oxygenase/FMN (oxyFMN) construct of human iNOS were determined by laser flash photolysis over the temperature range from 283 to 304 K. An appreciable increase in the rate constant value was observed with an increase in the temperature. Our previous viscosity study indicated that the IET process is conformationally gated, and Eyring equation was thus used to analyze the temperature dependence data. The obtained magnitude of activation entropy for the IET in the oxyFMN construct is only one-fifth of that for the holoenzyme. This indicates that the FMN domain in the holoenzyme needs to sample more conformations before the IET takes place, and that the FMN domain in the oxyFMN construct is better poised for efficient IET.

1. Introduction

Nitric oxide (NO) is one of the most studied small molecules in biology due to its involvement in numerous biological processes such as vasodilation, neurotransmission and immune response [1,2]. Mammalian NO synthase (NOS) is a homodimeric flavo-hemoprotein that catalyzes the conversion of L-arginine (Arg) to NO with NADPH and O₂ as co-substrates [3,4]. There is still much unknown about how NO production by NOS is tightly regulated [4-6]. It is of biomedical importance to study mechanisms of NOS regulation because unregulated NO production by NOS has been implicated in an increasing number of human pathologies, including cancer and ischemic injury caused by stroke [2,7].

There are three mammalian NOS isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Each NOS subunit comprises of an N-terminal oxygenase domain (containing a catalytic heme active site) and a C-terminal reductase domain (containing FAD and FMN cofactors), with a calmodulin (CaM) binding region between the two domains [4,5]. The substrate, L-Arg, and a cofactor, (6R)-5,6,7,8-tetrahydrobiopterin (H₄B), both bind near the heme center in the oxygenase domain. The iNOS isoform binds CaM tightly while nNOS and eNOS bind CaM reversibly in response to intracellular Ca²⁺ concentration [3,4]. The interdomain (intraprotein) electron transfer (IET) processes are key steps in NO synthesis [3,4,8,9]. Specifically, the CaM-controlled intersubunit FMN–heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain [10]. A laser flash photolysis approach, recently developed in our laboratories [11], has been used for direct determination of kinetics of the IET between catalytically significant redox couples of FMN and heme in bi-domain oxygenase/FMN (oxyFMN) constructs [11-13] and full-length NOS enzymes [13,14].

It is generally accepted that CaM-binding has little effect on the thermodynamics of redox processes in NOS [15-17], implying dynamic CaM regulation of IET via redox-linked conformational changes. A FMN-domain tethered shuttle model (Figure 1) was recently proposed [18] and supported by kinetics [12,14,19,20] and thermodynamic [21] studies. This model involves the swinging of the FMN domain from its original electron-accepting (input) state to a new electron-donating (output) state. The putative output state (bottom panel of Figure 1) is a complex between the oxygenase and FMN domains, which favors electron output from FMN to heme, and hence activates NO production. The output state structure has not yet been elucidated. Truncated two-domain NOS oxyFMN construct is a valid model of the output state [11,12], which only consists of the heme-containing oxygenase and FMN domains, along with the CaM-binding region [22]. This construct is a minimal electron transfer complex designed to favor the interactions between the FMN and heme domains [22].

Figure 1.

Tethered shuttle model. Flavodoxin-type FMN-binding domain (yellow) shuttles between the flavodoxin-NADPH reductase (FNR)-type “dehydrogenase unit” and the heme-containing oxygenase domain. Top: input state; bottom: output state. Free FMN domain conformations also exist in between the two docked states.

An appreciable decrease in the FMN–heme IET rate constant value of a human iNOS oxyFMN construct was observed with an increase in the solution viscosity [23]. The kinetics and NOS flavin fluorescence results indicate that the FMN–heme IET in iNOS is gated by a large conformational change, and that the docked FMN/heme state is populated transiently [23]. In the present work, we have investigated the temperature dependence of kinetics of the FMN–heme IET in truncated oxyFMN and full length human iNOS proteins. To our knowledge, this is the first temperature-dependence study of the NOS FMN–heme IET kinetics, which allows us to directly compare the temperature-dependent behavior of the IET process in the two protein systems.

2. Materials and Methods

Expression and purification of human iNOS oxyFMN and holoenzyme

The full length and oxyFMN human iNOS vectors and CaM expression vector were generous gift from Dr. Guy Guillemette (University of Waterloo, Canada). The iNOS plasmid was co-transfected with CaM expression vector (p209) into E. coli BL21(DE3) cells by electroporation (MicroPulser, Bio-Rad). The iNOS enzyme must be co-expressed with CaM in vitro because of its tendency to aggregate when residues of the highly hydrophobic CaM-binding region are exposed to an aqueous environment. Expression and purification of the human iNOS proteins was performed as previously described [13]. CaM binds tightly to iNOS and co-exists in the purified iNOS proteins.

Laser flash photolysis

CO photolysis experiments were conducted using an Edinburgh LP920 laser flash photolysis spectrometer, in combination with a Q-switched Continuum Surelite I-10 Nd:YAG laser and a Continuum Surelite OPO. A 446 nm laser pulse (out of the OPO module) was focused onto the sample cell to trigger the IET reactions. A 50 W halogen lamp was used as the light source for measuring the kinetics at ms – s time scales. A LVF-HL filter (Ocean Optics, FL) with band pass peaked at selected wavelength (580 or 465 nm) was placed before the partially reduced protein sample to protect it from photo-bleaching and further photo-reduction by the white monitor beam [9]. The sample temperature was controlled by using a TLC 50 cuvette holder coupled with a TC 125 temperature-controller (Quantum Northwest). Dry nitrogen gas was purged over the cuvette surface to avoid moisture buildup at lower temperature.

The CO photolysis experiments were performed as previously described [11,12,14]. Briefly, a CO/Ar (v/v ~ 1:3) pre-degassed iNOS solution was illuminated for a certain period of time to obtain a partially reduced form of [Fe(II)-CO][FMNH^•]. The sample was subsequently flashed with 446 nm laser excitation to trigger the FMN-heme IET, which can be followed by the loss of absorbance of FMNH^• at 580 nm, and the loss of absorbance of Fe(II) at 465 nm [13]. All the experiments were repeated at least twice. The transient absorbance changes were averaged and analyzed using OriginPro 8.5 (OriginLab).

3. Results and Discussions

3.1. The FMN–heme IET kinetics in human iNOS oxyFMN as a function of temperature

As expected, upon a 446 nm laser excitation, the absorption at 580 nm of the partially reduced human iNOS oxyFMN at 15 °C decays rapidly below the pre-flash baseline (Figure 2), which is due to the FMN–heme IET (eq 1, where FMN_hq stands for FMN hydroquinone), resulting in FMNH^• depletion [13], with a rate constant of 252.8 ± 2.2 s⁻¹.

$$\left[{\text{Fe}}^{\text{II}}\right]\left[{\text{FMNH}}^{•}\right]\rightleftharpoons \left[{\text{Fe}}^{\text{III}}\right]\left[{\text{FMN}}_{\text{hq}}\right]$$ eq 1

This is followed by a much slower recovery toward baseline (apparent rate constant = 2.5 ± 0.1 s⁻¹; Figure S1 in the Supporting Information), which is due to CO re-binding to Fe(II) [13]. Note the spectral “transition” (i.e. a reversal in direction of absorption changes over time) in the 580 nm traces.

Figure 2.

Transient trace at 580 nm at 0 – 0.04 s obtained for [Fe(II)–CO][FMNH^•] form of the human iNOS oxyFMN construct flashed by 446 nm laser excitation. The sample temperature was set at 15 °C. Red trace stands for the best fit using a single-exponential decay model. Anaerobic solutions contained 10 μM iNOS oxyFMN, ~ 20 μM dRF and 5 mM fresh semicarbazide in a 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 IET kinetics of the oxyFMN construct was determined over the temperature range from 283 to 304 K, and the rate constants k_et are listed in Table S1 in the Supporting Information. Note that an appreciable increase in the IET rate constant value was observed with an increase in the temperature. Importantly, the obtained rate constant of the rapid decay (Figure 1) over the temperature range is independent of the signal amplitude (data not shown), i.e. reduced protein concentration, confirming an intra-protein process.

3.2. The FMN–heme IET kinetics in human iNOS holoenzyme as a function of temperature

We could hardly observe the spectral transition in the 580 nm traces for the holoenzyme with our laser flash photolysis apparatus. This is likely due to less sensitivity of the current setup (compared to a laser flash photolysis instrument in Tollin laboratory, which allowed us to observe the IET in the holoenzyme at 580 nm [13]). Another factor could be “interference” from FAD in the holoenzyme, which should not undergo IET under the experimental conditions. Owing to equipment sensitivity and background absorption of FAD in holo-iNOS, we need to switch to 465 nm where absorption of heme species dominates. Our previous studies clearly showed that the FMN-heme IET in iNOS can be also monitored by absorbance decrease at 465 nm (upon laser excitation), which is due to net oxidation of heme in eq 1 [13]; see Figure S2. Indeed, using our current laser flash photolysis apparatus, we obtained the same IET rate constants for the oxyFMN construct by measuring transient traces at 580 and 465 nm (data not shown). The 465 nm trace has larger amplitude (compared to the 580 nm trace), which is due to much larger absorption coefficients of the heme species than the flavin species in NOS [13]. This offers an alternative to determine the IET kinetics of NOS samples whose IET can be barely observed at 580 nm. It is important to note that ΔA₄₆₅ obtained for the holo-iNOS sample on our apparatus at room temperature of 21 °C gave an IET rate constant of 34.0 ±0.4 s⁻¹, which is in excellent agreement with the reported value (32 ± 3 s⁻¹) obtained for a different preparation of human iNOS holoenzyme by using another laser flash photolysis system [13].

We thus measured the IET kinetics in the holoenzyme by monitoring absorption change at 465 nm. Likewise, upon CO photolysis, the absorption at 465 nm of the partially reduced holoenzyme decays below the baseline with a rate constant of 17.2 ± 0.2 s⁻¹ (Figure 3), followed by a slower recovery toward baseline (Figure S3). The IET kinetics of human iNOS holoenzyme was also determined over the temperature range from 283 to 304 K, and the rate constants are listed in Table S1 in the Supporting Information.

Figure 3.

Transient trace at 465 nm at 0 – 0.4 s obtained for the [Fe(II)–CO][FMNH^•] form of the human iNOS holoenzyme flashed by 446 nm laser excitation. The sample temperature was set at 10 °C. Red trace stands for the best fit using a single-exponential decay model. Anaerobic solutions contained 15 μM iNOS holoenzyme, ~ 20 μM dRF and 5 mM fresh semicarbazide in a 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).

3.3. Fit of the temperature-dependence data

Our previous viscosity study indicated that the NOS FMN-heme IET process is conformationally gated [23]. The temperature dependence of the IET rate constants was thus analyzed by transition state theory using the Eyring equation,

$$\text{ln}(\text{k}∕\text{T})=-\Delta H^{‡}∕\text{RT}+\Delta S^{‡}∕\text{R}+\text{ln}({\text{k}}_{\text{B}}∕\text{h})$$ eq 2

where ΔH^‡ is the activation enthalpy, ΔS^‡ is the activation entropy, h is the Planck’s constant, k_B is the Boltzmann constant, and R is the gas constant. The obtained Eyring parameters are listed in Table S2. Temperature dependence behaviors of gated electron transfer processes in other proteins have been analyzed similarly [24,25].

The best fit of eq 2 to the oxyFMN data was realized with ΔH^‡ = 52.6 ± 3.7 kJ/mol and ΔS^‡= −17.1 ± 1.2 J/mol/K (Figure 4). For the holoenzyme, its activation enthalpy value (36.9 ± 1.6 kJ/mol) is a bit lower than that of oxyFMN, while its activation entropy value (−89.7 ± 4.5 J/mol/K) is about five times of the oxyFMN. This clearly indicates that the FMN domain in oxyFMN construct is better poised for efficient IET (which is consistent with the fact that the oxyFMN construct is designed to favor the interactions between the FMN and heme domains [22]). It is of note that the obtained activation values are comparable to those of inter-flavin electron transfer in human cytochrome P450 reductase [25], which is homologous to NOS reductase domain.

Figure 4.

Eyring plots showing the temperature dependence of k_et measured for oxyFMN (circle) and full length (triangle) human iNOS proteins. The obtained ΔH^‡ and ΔS^‡ values are listed in Table S2 in Supporting Information.

During NOS catalysis, the FMN domain cycles between interaction with an NADPH/FAD domain (to receive electrons) and interaction with an oxygenase domain (to deliver electrons to the NOS heme). In NOS holoenzyme, the FMN domain exists in two distinct docked states and other free states (Figure 1). Recent kinetics and fluorescence results suggested that the NADPH/FAD domain has a much greater capacity to interact with the FMN domain than does the oxygenase domain [26]. Therefore the majority state in the holoenzyme should be in the input state (probably because of the inter-domain FAD/FMN interactions), which must redistribute to an IET-reactive conformation (i.e. output state) before the IET takes place. The higher magnitude of the negative activation entropy (see above) is consistent with the FMN-heme IET being gated by motion of the FMN domain, which in the holoenzyme can sample more conformations (that are not IET-competent) than in the oxyFMN construct.

A conformational sampling model was recently proposed for the electron transfer (ET) P450 reductase protein [25], where sampling of a continuum of conformational states gives a range of transient donor-acceptor complexes, only a subset of which are ET-competent. We recently showed that the FMN–heme IET in iNOS is gated by conformational change of the FMN domain, and that the docked FMN/heme state is populated transiently [23]. The observed rate of heme reduction for a population of iNOS conformations can thus be limited by the relatively infrequent formation of the docked IET-competent complexes (when the rate of conformational change is comparable to that of the IET). Note that the NOS heme reduction needs to be slow enough to allow the enzyme to promptly release the NO that it makes [5]. This may explain why sampling more conformation would be “beneficial” to iNOS function, where iNOS holoenzyme would have a low-frequency formation of productive FMN/heme complexes, despite their bi-domain structure potentially enabling a much more efficient FMN-heme IET as occurs in the oxyFMN construct.

We are aware that this may not be a unique explanation of the temperature-dependence data. There are other possible explanations including changes in midpoint potentials of the redox factors, and change in the rate-limiting step for the IET [27]. Additionally, analyses of the data with Marcus theory [28] and thermodynamic data of the oxyFMN [22] and full length iNOS proteins [16] did not give unreasonable values of H_AB and IET distance (data not shown). Nonetheless, in combination of recent viscosity dependence data [23], our current gated-IET model represents a more reasonable explanation. These results need to be extended by future thermodynamic and dynamic studies, coupled with structural information. These experiments need considerable efforts and are outside of the scope of the present study.

In conclusion, an appreciable increase in the NOS FMN-heme IET rate constant value was observed with an increase in the temperature over the range of 283 – 304 K. Comparative studies of the temperature behavior of human iNOS oxyFMN and holoenzyme proteins, in combination with our previous viscosity dependence results [23], indicate that the FMN domain in the holoenzyme needs to sample more conformations before the IET takes place, and that the FMN domain in oxyFMN construct is better poised for efficient IET.

Research Highlights.

• The FMN–heme IET kinetics in full length and truncated oxygenase/FMN construct iNOS proteins were determined at 283-304 K.

• This is the first study of temperature dependence of the NOS IET kinetics.

• Eyring equation was used to analyze the temperature dependence data.

• The magnitude of activation entropy for the IET in the oxygenase/FMN construct is only one-fifth of that for the holoenzyme.

• The results indicate that the FMN domain in the holoenzyme needs to sample more conformations before the IET takes place.

Abbreviations

NO

nitric oxide

NOS

nitric oxide synthase

iNOS

inducible NOS

nNOS

neuronal NOS

eNOS

endothelial NOS

CaM

calmodulin

oxyFMN

bi-domain NOS construct in which only the heme-containing oxygenase and FMN domains along with the CaM binding region are present

FMNH^•

FMN semiquinone

FMN_hq

FMN hydroquinone

FAD

flavin adenine dinucleotide

IET

intraprotein electron transfer

ET

electron transfer

k_et

rate constant for electron transfer

dRF

5-deazariboflavin

H₄B

(6R)-5,6,7,8-tetrahydrobiopterin