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Oxygen activation in NO synthases: evidence for a direct role of the substrate

Nitric oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho‐physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast‐kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise FeII‐O2 reaction intermediates in the presence of L‐arginine (Arg), alkyl‐ and aryl‐guanidines. The presence of new reaction intermediates, such as ferric haem‐peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H‐bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (Nω‐hydroxy‐L‐arginine oxidation).

Nitric oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho-physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast-kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise Fe II -O 2 reaction intermediates in the presence of L-arginine (Arg), alkyland aryl-guanidines. The presence of new reaction intermediates, such as ferric haem-peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H-bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (N x -hydroxy-L-arginine oxidation).
Nitric oxide (NO) synthases were first characterised in mammals in the early 1990s as the source of NO in mammalian cells. Constitutive NO synthases (NOSs) are involved in signalling processes such as blood pressure regulation and angiogenesis (eNOS) [1] or learning process, synaptic plasticity and neurotransmission (nNOS) [2]. The inducible NOS (iNOS) is recruited for nonspecific immune response against tumours, viruses or bacteria [3]. On the other hand, NOSs have been increasingly associated with the development of several pathologies including cardiovascular, inflammatory and neurodegenerative diseases, diabetes and cancers [4][5][6][7].
Despite a broad range of biological activities, mammalian NOSs share a similar 3D structure and a common catalytic mechanism. NOSs synthesise NO in two stages [8]: the first haem-based oxidation converts Larginine (Arg) to a stable intermediate, N x -hydroxy-Larginine (NOHA), and the second converts NOHA to L-citrulline and NO [9,10]. Mammalian NOSs are homodimers that contain an N-terminal oxygenase domain (NOSoxy) and a C-terminal reductase domain (NOSred). Electron transfer between the two domains is regulated by a Calmoduline-binding interface. NOSoxy binds Arg, the haem cofactor and the redox-active cofactor tetrahydrobiopterin (BH 4 ). NOSred is similar to P450 reductase and thus contains an NADP(H)-and FAD-binding flavodoxin-reductase module and a flavodoxin-like FMN-binding domain [8,11,12]. Due to the high biomedical importance of mNOSs, their molecular mechanism has attracted tremendous interest. The first O 2 activation step (Step 1) is similar but distinct from that of the cytochromes P450 ( Fig. 1) [13]: NOS catalysis begins with the reduction in the high-spin ferric haem (Fe III ) to ferrous haem (Fe II ) by NOSred (with electrons originating from NADPH). Subsequent O 2 binding forms the ferric superoxo-haem (Fe III -OO°) [14], which is further reduced by BH 4 and protonated to form the ferric hydroperoxo-haem (Fe III -OOH) [15][16][17][18] [20]. However, this sequence remains essentially hypothetical. The only reaction intermediate that has been identified so far, besides the oxygenated complex, is the haem-peroxide species, and only through cryoreduction approaches [21,22] that do not mimic catalytic conditions. There is also no clear understanding of the reason of the coexistence of two distinct mechanisms for the two catalytic steps: if the first step of O 2 activation is similar to what has been proposed for cytochromes P450, the second step is specific to NOS. Many research programmes have investigated two major specificities of NOS catalysis, which is the electron transfer by the BH 4 cofactor [23][24][25] and the push effect of the proximal ligand [26][27][28]. However, only scarce attention has been devoted to the proton transfer process (nature of the proton donor, sequence and pathway of proton transfer). Proton transfer is also at Fig. 1. Catalytic and molecular mechanism of NOS. A: The two oxidative steps that sequentially convert the guanidinium moiety of Arg into NOHA and L-citrulline with NO as side-product. B: Current model of NO-Synthase molecular mechanism. So far, only the initial and final intermediates (Fe II -O 2 and Fe III -NO/Fe III ) have been trapped and characterised in catalytic conditions. The key catalytic intermediates (haem-peroxo, oxoferryl and ferrous haem-NO complexes) remain yet to be identified. the heart of NOS molecular mechanism for it controls the balance between Arg hydroxylation (two protons) and NOHA oxidation (one proton), and tunes the coupling between electron transfer and oxygen activation, preventing NOS uncoupling and ROS production.
There are only few data on proton transfer and no comparative investigation of Steps 1 and 2. This is mostly due to the instability of the Fe II -O 2 intermediate, which requires often the use of noncatalytic conditions (redox analogues, low temperature. . .). However, some reports have addressed the question of proton transfer and/or the protonation of haem-oxo species [13]: among them, a large series of works have established the influence of the distal environment on the electronic structure, stability and reactivity of Fe II -O 2 , -CO and -NO species [29][30][31][32][33]. X-ray and Raman data [29,32,[34][35][36] have clearly shown that the H-bond distal network undergoes a rearrangement as a function of the nature of the substrate (Arg, NOHA, analogues), with changes in the interaction between the substrate guanidinium and the distal ligand and in the role of a vicinal water molecule. We utilised a series of Arg analogues that were distributed along three groups according to their influence on the distal H-bond network as determined from the spectroscopic signature of the CO complex probed with resonance Raman (RR) spectroscopy [33]: Family 1 analogues had an effect similar to that of L-Arg, Family 2 similar to NOHA and Family 3 corresponded to analogues that bind the active site but do not induce a spectroscopic signature, thus mimicking the absence of substrate. We showed that these changes modify the reactivity of the Fe II -O 2 species [33,37] and the fate of the ferric haemperoxo intermediate: in the presence of Arg, the oxo ligand would be H-bonded to the water molecule, favouring the O-O heterolytic cleavage (and the buildup of a Cpd I-like species), whereas the direct H-bond between NOHA guanidinium and the peroxo species would favour its stability and its direct reactivity [33]. This indicates that the difference between Steps 1 and 2 depends on the structure of the distal H-bond network: the distal water molecule would therefore be required for the first catalytic step (Arg hydroxylation) but not for the second one (NOHA oxidation) [13,33]. This has been confirmed by the work of Marletta and coworkers with 5-methyl Arg and NOHA analogues [38].
This model has been mostly thought of from data using Fe II -O 2 mimics such as the stable Fe II -CO and Fe II -NO complexes. However, these species present structural and electronic properties different from those of Fe II -O 2 . To overcome the instability of Fe II -O 2 , that prevents its direct investigation, we have used here two complementary fast-kinetics approaches: (a) the stopped-flow set-up coupled to UV-vis spectrometry allows determining the rate constants of O 2 activation and autoxidation; (b) the T-mixer set-up coupled to RR spectroscopy allows characterising the structural properties of Fe II -O 2 intermediate and identifying short-lived intermediates undetected by UV-visible spectroscopy.
We used this approach on one hand to investigate the effect of Arg analogues on the Fe II -O 2 complex and verify the actual role of the water molecule in NOS catalysis, and on the other hand to trap and characterise new reaction intermediates that may intervene in Arg hydroxylation.

Enzyme preparation
Mouse iNOS oxygenase domain (iNOSoxy) containing a six-histidine tag at its C-terminus was expressed in Escherichia coli BL21 using the PCWori vector and purified as already described [39] in the absence of BH 4 and Arg.

Stopped-flow experiments
The protein samples containing BH 4 /BH 2 were obtained by preincubation at 4°C in the presence of 200 lM BH 4 / 400 lM BH 2 and 5 mM Arg or 10 mM of compounds Pentylgua and 4-Fphgua in 100 mM Kpi buffer (pH 7.4). The binding of the guanidine derivatives and BH 4 was confirmed by measuring the low-spin (420 nm) to the high-spin (395 nm) transition of the haem using UV-vis spectroscopy. The samples were made anaerobic by several cycles of vacuum and argon refilling. The reduction in the haem was achieved by progressive addition of an anaerobic solution of sodium dithionite and followed by monitoring the change in the Soret peak from 395 nm for the ferric form to 412 nm for the ferrous one. The rapid-mixing stopped-flow experiments were performed at 4°C on a Bio-Logic SFM 300 instrument customised for anaerobic and semianaerobic experiments and connected to a Tidas 1024diode array detector able of recording spectra every 3 ms. The reduced enzyme was transferred into the stopped-flow apparatus with a gastight syringe and was rapidly mixed with an equal volume of air saturated buffer. Spectra were recorded between 350 and 700 nm with a total measurement time between 2 and 5 s.

Stopped-flow data analysis
We used a direct examination of the recorded spectra with the absorbance cross sections at specific wavelengths performed with ORIGINPRO 8.0 software (OriginLab Corporation, Northampton, MA, USA). First, visual analysis of the spectra with BH 2 allowed us to identify the different haem species involved in the single-turnover reaction: ferrous Fe II (412 nm), ferrous dioxygen Fe II -O 2 (427-430 nm) and ferric Fe III (395 nm) complexes.
The determination of quantitative transition rates were obtained by combining different methods. The autoxidation rates (in the presence of BH 2 ) reported by Moreau et al. [37] were determined by global analysis (SPECFIT) and by simulation of time traces at 428/395/650 nm to a mono-exponential function. Rate constants obtained by these different methods were similar for every condition [37]. In this report, autoxidation rates were obtained by global analysis like Moreau et al. [37] but also by simulation of the 428 nm time trace to a biexponential function. Here again, rates obtained via these two methods were similar.
Activation rates (in the presence of BH 4 ) were all obtained like Moreau et al. [37], that is, by simulation of the 395 nm time traces to a single mono-exponential function, except in the presence of Arg and 4-methoxyphenylguanidine, for which we simulated the kinetics to a biexponential function.

Resonance Raman spectroscopy of iNOSoxy oxygenated intermediates
The buffer used for kinetic measurements was 100 mM KPi, pH 7.4. To prepare the ferrous form of iNOSoxy, the ferric enzyme (80 lM) was equilibrated with nitrogen gas for 30 min at room temperature, and the haem was then reduced with sodium dithionite. Complete reduction in iNOSoxy was verified by optical spectroscopy. Oxygen-containing buffer solutions were prepared by equilibrating deoxygenated buffer with 16 O 2 or 18 O 2 . The rapid T-mixer used here was described previously [40]. Oxygen was removed from the mixer with an anaerobic buffer prior to connecting the syringes containing the ferrous protein and oxygen-saturated buffer. The output at 413.1 nm is from a Kr ion laser (Innova 302 Kr; Coherent, Santa Clara, CA, USA) at 10 mW. The output at 441.6 nm is from a He/Cd ion laser (Liconix Laser; Melles-Griot, Ottawa, ON, Canada) at 10 mW. The position of the laser-focusing point was moved along the flow direction to obtain the desired time point. The flow rate was adjusted to result in a time point of 1.25 ms mm À1 . The data were measured at time delays after mixing ranging from 4 to 40 ms and measurements were recorded at room temperature (25°C). The RR spectra were calibrated with the signals from indene. The RR spectrum of reduced myoglobin was recorded prior to each mixing experiment and was used to adjust small differences in the calibration of spectra from different mixing experiments.

Kinetic analysis of Fe II -O 2 activation and autoxidation reactions
We first investigated the effect of Arg and various aryl-guanidines on the kinetics of oxygen activation and of Fe II -O 2 autoxidation. This work aimed at completing the results we previously obtained [33,37] for supplemental compounds of Family 2 and 3 analogues ( Fig. 2) in order to determine the best conditions for the T-mixing experiments observed by RR spectroscopy, in particular the optimal build-up times. We used the same stopped-flow methods and the same kinetic analysis [33,37] (see Experimental procedures) to determine the rates of Fe II -O 2 autoxidation in the presence of BH 2 (a redox-inactive BH 4 analogue) and the rates of oxygen activation, in the presence of BH 4 .
In the presence of BH 2 , we observed the same Fe II ? Fe II -O 2 ? Fe III transition in the presence of Arg (Fig. 3A) or various aryl-guanidines analogues (Fig. 3B). The rates of Fe II -O 2 decay were determined by simulating the time traces at 427 nm (Fe II -O 2 formation and decay) and at 395 nm (Fe III recovery) with a biexponential function (see Experimental procedures). Our results confirm that changes in the substrate does not modify oxygen-binding rate (k 1 ) but that autoxidation rates (k 2 ) successively increase for Family 1 and Family 2/3 analogues, as already described [37] (Fig. 4A, Table 1). In the presence of BH 4 , the same sequence of intermediates is observed (Fig. 4B) with a smaller build-up of the Fe II -O 2 species (Fig. 3C,D), illustrating the oxygen activation upon an electron transfer from BH 4 , as already reported [41][42][43]. The kinetics analysis confirms the absence of Fe II -O 2 buildup in the presence of aryl-guanidines analogues, as previously reported [41][42][43]. The moderate increase in the rate of Fe III recovery (between 12% and 100%, Table 1) should not have prevented observing the build-up of this intermediate (Fig. 4C), suggesting the existence of a transient reaction intermediate that cannot be observed by conventional UV-visible spectrometry, as already proposed [37].
Our results confirm the existence of three families of aryl-guanidine that do not modify binding and coordination of dioxygen to iNOSoxy but that might affect Fe II -O 2 stability [37]. Kinetic analysis of oxygen activation in the presence of BH 4 supports our early hypothe-sis of the transient formation of an additional reaction intermediate [37]. We then used RR spectroscopy coupled to fast-kinetics methods to directly analyse the effect of aryl-guanidine binding on dioxygen coordination and to look for potential new intermediates. L-Arginine N ω -Hydroxy-L-Arginine n-Pentyl-guanidine Aryl-guanidines X = F 4-Fphgua CF 3 4-CF 3 -phgua Cl 4-Clphgua OCH 3 Fphgua (B, D). The enzyme was rapidly mixed at 4°C with an equal volume of oxygen-saturated buffer (see Experimental procedures). Solid lines correspond to the spectra of the initial reduced forms; dashed and dotted lines correspond to the spectra at various times after rapid-mixing.

Resonance Raman spectroscopy characterisation of NOS mechanism of hydroxylation
The nature of the intermediates formed in the course of Arg and Arg analogues oxidation have been analysed by resonance Raman spectroscopy using a homebuilt T-mixer set-up [40] (see Experimental procedures). Solutions of reduced iNOSoxy in the presence of Arg or Arg analogues are mixed with 16 O 2 -or 18 O 2saturated buffers and continuously pushed along a 250-lm wide quartz tube. Focusing the RR laser beam at different distances from the mixing point allows determining the vibration modes of all species present in the reaction sample at a defined time (see Experimental procedures). We used two excitation wavelengths (413.1 and 441.6 nm) and recorded a spectral window that covers the 650-1600 cm À1 region. We focused on characteristic porphyrin modes and looked for the appearance of new bands that could reflect the O-O stretch modes of Fe II -O 2 or other haem-peroxo or hydroperoxo complexes. We used the kinetics data obtained by stopped-flow to define the time of maximal Fe II -O 2 build-up (see Experimental procedures).
We first look to iNOSoxy-catalysed oxidation of Arg (Fig. 5). The upper spectrum (Fig. 5A) shows the RR fingerprint of the sample for a 10 ms reaction time in the presence of 16 O 2 . We observe a mixture of species with a minor fraction of the initial Fe II species (bands m 4 at 1469 cm À1 ) and mostly ferric-like complexes with characteristic bands at m 7 (677 cm À1 ), m 5 (1128 cm À1 ) and m 4 (1373 cm À1 ). Most of iNOSoxy seems to have reacted to form a mixture of Fe III and Fe II -O 2 species with characteristic m 3 bands at 1488 cm À1 (Fe III ) and 1502 cm À1 (Fe II -O 2 ) [29,32]. The Fe II -O 2 m O-O is difficult to detect for it is expected around 1125 cm À1 [32] and is overlapping with the porphyrine mode m 5 (1122 cm À1 ). For this reason, we achieved the same experiment with a 18 O 2 -saturated buffer that results in an isotopic shift of m O-O down to 1060 cm À1 but leaves m 5 unaffected (Fig. 5B). The difference between the 16 O 2 and the 18 O 2 spectra (Fig. 5C)  the reaction sampled at 10 ms are displayed in Fig. 6. We observed porphyrin modes of the ferric species (Fig. 6, spectra A Table 2) The last series of T-mixer/RR experiment was achieved in the presence of BH 4 and 4-Fphgua, an Arg analogue from Family 2 for which no Fe II O 2 intermediate has been observed by stopped-flow [33]. We observed the final ferric iNOSoxy modes at 677 cm À1 (m 7 ), 1122 cm À1 (m 5 ),1374 cm À1 (m 4 ) and 1488 cm À1 (m 3 ) with again a contribution at 1506 cm À1 that could be assigned to the m 3 mode of an Fe II O 2 intermediate (Fig. 7, spectra A, B). The 16 O 2 / 18 O 2 difference spectrum (Fig. 7, spectrum C) reveals the presence of m O-O vibrational mode in both the 16 O 2 and 18 O 2 conditions. The fitting of the 1000-1200 spectral region (Fig. 7, inset) leads to m O-O frequencies at 1130 ( 16 O 2 ) and 1064 cm À1 ( 18 O 2 ). Unexpectedly, these modes were not observed using an excitation wavelength at 441.6 nm and did not allow either to identify other haem-oxy intermediates (data not shown).

Discussion
The characteristics of proton transfer process are a major topic to understand the mechanism of oxygen activation by NOS. Using a series of synthetic analogues of Arg, we recently proposed a model with the distal water molecule as potential second proton donor [33]. The H-bond established between this water molecule  and the dioxygen ligand would be stabilized in the presence of Arg, but not NOHA. Thus, the nature of the distal H-bond network appears as a key element in tuning ferric haem-superoxo/peroxo reactivity and as such might control the balance between heterolytic cleavage (Step 1) and nucleophilic attack (Step 2) [13,33,36].

A new approach to investigate oxygen activation mechanism
In this work, we wished to extend the model we deduced from the analysis of the structure of the Fe II -CO complex [33] to the relevant Fe II -O 2 intermediate and establish a direct correlation between Fe II -O 2 structure/reactivity and its interaction with the distal H-bond network. Following our first hypotheses [37], we aimed at identifying potential new haem-oxy intermediates formed in the course of aryl-guanidines oxidation. For this matter, we chose to combine the use of synthesised Arg analogues to an alternative and efficient fast-kinetic approach, a T-mixer device coupled to RR spectroscopy [40]. Continuous flow analysis allows recording RR spectra for reaction times as small as a millisecond. These short-time recordings  might allow identifying short-lived intermediates such as the Fe II -O 2 complex in the presence of Arg analogues. Besides, resonance Raman is an appropriate spectroscopy for characterising Fe II -O 2 structural properties and for identifying species that cannot be observed by UV-visible spectroscopy. We first furthered our initial stopped-flow experiments in order to complete the set of kinetic parameters for single-turnover oxidation reactions for various analogues of Arg (Table 1). Monitoring the absorbance at 428 nm during stopped-flow analyses allows the estimation of the Fe II -O 2 build-up and decay kinetics for the various oxidation reactions. It provides kinetic information that is essential to the trapping of reaction intermediates. We used two distinct excitation wavelengths  [45]. For this reason, comparison among the analogues and substrates are discussed based on the 18 O 2 frequencies. In the presence of Pentylgua, the m O-O frequency increases by 1-2 cm À1 (2-4 cm À1 in 16 O 2 conditions), which is reminiscent to what has been observed for the m C-O mode of the Fe II -CO complex [33], suggesting a weakening of the H-bond between the water molecule and the dioxygen ligand. Although the arginine ? Pentylgua substitution preserves the structure of the H-bond (and the H-bonding with the water molecule) changes in the guanidine structure might lead to a small destabilisation and weakening of this H-bond, which is illustrated by the increased Fe II -O 2 autoxidation rate in the presence of Pentylgua. On the other hand, the Arg analogues from Family 2 are believed to modify the H-bond distal network in a way similar to that observed for NOHA, that is, by displacing the water molecule away [33]. This led to a strengthening of Fe II -CO m C-O stretch [33] Table 2). The same effect is observed in the presence of 4-Fphgua, an aryl-guanidine from Arg analogue Family 2, with an increase in 4 cm À1 of the m O-O frequency. This supports our hypothesis concerning the effect of aryl-guanidines on the distal H-bond network: the pKa of these aryl-guanidines (Family 2: 4-Fphgua, 4-CF 3 -phgua, and 4-Clphgua) is weaker than that of L-Arg [33]. This gives to the guanidinium proton a more acidic character, which in turn modifies the H-bond network in such a way that the water molecule does not interact anymore with the ligand bound to the haem iron.
Our results show that despite its more linear coordination, CO remains a relatively good mimic of O 2 to investigate O 2 interactions with distal residues and reactivity. Globally, our results confirmed the correlation between the evolution of the vibration modes of the CO and O 2 ligands that sense the changes in the distal environment and in the intensity of the electron back-donation from iron orbitals. The role of NOS substrate in the proton transfer pathway and the nature of proton donor remains a matter of debate. Various reports from the groups of Rousseau [32], Crane [36], Marletta [38], Poulos [35] and from our groups [33,37] support a model of NOS-catalysed oxygen activation in which neither Arg nor NOHA directly act as the second proton donor. Instead, Arg would stabilise the distal H-bond network in which the water plays a crucial role in the proton transfer sequence and in the formation of the oxoferryl intermediate (Step 1). In the presence of NOHA, the removal of the water molecule and the H-bond between the distal oxygen and the substrate guanidinium stabilises the peroxo intermediate and prevents the Cpd I formation.

Looking for new reaction intermediates
The second axis of our project was to look for reaction intermediates in NOS-catalysed oxygen activation, using the T-mixer/RR set-up to reveal new species, in particular ferric haem-peroxide that could not have been observed by UV-visible absorption spectroscopy. The initial hypothesis rose from the absence of Fe II -O 2 intermediate build-up in the presence of BH 4 and Family 2 aryl-guanidines [37]. This was in opposition with the observation of Fe II -O 2 build-up, (a) in the presence of BH 2 and (b) in the presence of BH 4 and Family 3 analogues, both conditions for which no electron transfer is achieved [37]. This suggests that Family 2 aryl-guanidines do not prevent O 2 binding. The fast Fe II -O 2 build-up opposed to the slow Fe III recovery, suggests the existence of an additional fast step converting Fe II -O 2 into an unknown intermediate that would slowly decay into the final Fe III species (Fig. 4C). Since we were not able to identify this intermediate by stopped-flow, we used the T-Mixer/RR setup to trap and characterise it.
We were able to observe the Fe II -O 2 species in (almost) all conditions. We did not detect additional porphyrin or m O-O bands, which suggested the absence of additional haem intermediate in the presence of Arg and Pentylgua (Family 1), but also with 4-Fphgua, for which we previously hypothesised the transient formation of a haem-peroxide complex. The nonobservation of bands specific to haem-peroxide complex in the crowded 750-800 cm À1 spectral region might, however, be due to a weak build-up of these intermediates (that would rapidly decompose into ferric species) and to the weak amplitude of their m O-O bands.
The question of the absence of Fe II -O 2 build-up in stopped-flow experiment remains, leading to two alternative hypotheses: (a) the absorbance of iNOSoxy in the presence of BH 4 and 4-Fphgua might be blueshifted with an absorption maximum around 420 nm, such as what has been described for Oxy 1 species, a CYP-like ferrous haem-oxo species [30,31]. This is supported by the absence of RR exaltation of the Fe II -O 2 modes using an excitation wavelength at 441.6 nm, which also suggest a blue-shift of Fe II -O 2 absorption. This shift would make the detection of Fe II -O 2 more difficult by optical absorption spectroscopy due to an absorption maximum too close to the Fe II and Fe III species. However, the BH 2 /4-Fphgua Fe II -O 2 species displays a maximum absorption around 428 nm, and Family 3 aryl-guanidines do not seem to modify this spectroscopic fingerprint. (b) The intermediate is not a haem-oxy but a low-spin ferric species with an absorption maximum around 415-420 nm. This intermediate could correspond to the produced hydroxy-aryl-guanidine that would be formed rapidly but would remain O-bound to the haem iron [46,47], which resulted in a Low-Spin signature. It would be released slowly, with a rate corresponding to the ferric Low-Spin/High-Spin transition (Fig. 4B). However, our T-Mixer/RR experiments did not show any low-spin signals (apart from that assigned to Fe II -O 2 ) that could suggest the presence of such a Fe III LS intermediate but instead reported the formation of a Fe II -O 2 species. At this stage, further experiments at longer reaction times are needed to favour any of these hypotheses.

Conclusion
In this work we combined the organic synthesis of tailored substrate analogues with a fast-kinetic approach that coupled a T-Mixer module to resonance Raman spectroscopy: the T-mixer allows investigating of short reaction times and RR is an alternative to optical spectroscopy for deciphering the nature of transient intermediates species. The experiments presented here allowed us to complete the structural characterisation of the Fe II -O 2 intermediate in the presence of various aryl-and alkyl-substrate analogues. However, this advanced approach failed in identifying new intermediates in the oxygen activation reaction of NO-Synthases. The peculiar absence of Fe II -O 2 build-up in the presence of various aryl-guanidines Arg analogues by optical spectroscopy remains unexplained so far and the proposed ferric haem-peroxide species elude any characterisation. Nonetheless, our results confirm the role of the H-bond network, in particular the acidity of the guanidinium and the interaction with the vicinal water molecule. This strengthens the scheme proposed for proton donation [13] that explains the difference between the first catalytic step (the water molecule allows the transfer of two protons and the formation of a [Fe III -OOH 2 ] + intermediate) and the second catalytic step (no water molecule, one proton transfer leading to a Fe III -OOH intermediate). This supports the model proposed for NOS molecular mechanism, for which Arg hydroxylation is related to the heterolytic cleavage of the [Fe III O-OH 2 ] + bond and the build-up of a Cpd I species unlike NOHA oxidation for which the Fe III -OOH species is the oxidative intermediate.