The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

Brunel, Albane; Wilson, Adjélé; Henry, Laura; Dorlet, Pierre; Santolini, Jérôme

doi:10.1074/jbc.m110.195446

Cited by 20 publications

(25 citation statements)

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“…Thus, the Phe mutants show increased reactivity of their Fe II -NO and Fe II -O 2 complexes. In summary, from the nNOSoxy results, it appears that an increased basicity of the proximal ligand can induce a greater push effect and a higher Fe II -NO reactivity, consistent with other observations in different NOS proteins (27)(28)(29)(30)(31). We observe a loss of the thiolate bond upon NO binding associated with mutants with very low heme midpoint potentials.…”

Section: The Proximal Cysteine Environment and K Ox Ratessupporting

confidence: 90%

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases

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Santolini

et al. 2019

Journal of Biological Chemistry

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Edited by F. Peter Guengerich Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from L-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main ratelimiting steps. These are the rate of heme reduction by the flavin domain (k r), of dissociation of NO from the ferric heme-NO complex (k d), and of oxidation of the ferrous heme-NO complex (k ox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to return to the catalytic cycle, and thus, enables a steady-state NO synthesis rate. Previously, we have reported that this reaction does involve the reaction of oxygen with the NO-bound ferrous heme complex, but the mechanistic details of the reaction, that could proceed via either an inner-sphere or an outersphere mechanism, remained unclear. Here, we present additional experiments with neuronal NOS (nNOS) and inducible NOS (iNOS) variants (nNOS W409F and iNOS K82A and V346I) and computational methods to study how changes in heme access and electronics affect the reaction. Our results support an inner-sphere mechanism and indicate that the particular heme-thiolate environment of the NOS enzymes can stabilize an N-bound Fe III-N(O)OO ؊ intermediate species and thereby catalyze this reaction, which otherwise is not observed or favorable in proteins like globins that contain a histidinecoordinated heme. This article contains Tables S1-S3 and Figs. S1-S7.

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Section: The Proximal Cysteine Environment and K Ox Ratessupporting

confidence: 90%

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases

Tejero

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Santolini

et al. 2019

Journal of Biological Chemistry

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“…This again emphasizes the fact that the hydrogen bonds to the proximal Cys ligand in the hs ferric state of {heme-thiolate} active sites have only a minor effect on the absolute strength of the Fe–S bond. This is in agreement with the resonance Raman results (see “Introduction”), which also show small shifts of only a few wavenumbers in the Fe(II)–CO stretches of the ferrous CO adducts in the W409 variants [39]. In the case of nNOSoxy, studies on the ferric NO adduct show the Fe–NO stretch at 546 cm −1 [33].…”

Section: Summary and Implicationssupporting

confidence: 88%

“…This conclusion is based on structural data and the fact that the imidazole ring has a lower p K a than the indole N–H group, which makes the former a better H-bond donor [39]. This stronger H-bond in W409H should lead to a reduction of the Fe(III)–S(Cys) bond strength, but the magnitude of this effect is not clear [23].…”

Section: Resultsmentioning

confidence: 99%

“…Couture and coworkers studied the W to F, Y, and H mutants of bacterial NOS from Staphylococcus aureus (where the F and Y mutants eliminate the hydrogen bond to Cys (see “Results and Discussion”), whereas for the H mutant, the hydrogen bond is presumably stronger [36]), and found that although these mutants alter the redox potential of the heme by over 100 mV, the effect on the Fe–CO stretching mode in the low-spin ferrous form of the enzyme is very small (±2–3 cm −1 relative to wt) [37]. A similar set of mutants was investigated for bacterial NOS [38] from Bacillus subtilis , again leading to the observation of very similar Fe–CO stretching frequencies with ν (Fe-CO) = 501 cm −1 in wt enzyme, and ν (Fe-CO) = 500, 501.5 and 504.5 cm −1 in W66F, W66Y and W66H, respectively [39]. Similar studies on the ferric NO complexes of wt rat nNOSoxy and the W to F and Y mutants have also been performed [33].…”

Section: Introductionmentioning

confidence: 99%

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Exploring second coordination sphere effects in nitric oxide synthase

et al. 2016

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Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the ‘Cys pocket’. These hydrogen bonding networks are generally believed to regulate the Fe–S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm−1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe–S(Cys) bond strength, at least in the hs ferric form of the protein studied here.

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“…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 and the push effect of the proximal ligand . However, only scarce attention has been devoted to the proton transfer process (nature of the proton donor, sequence and pathway of proton transfer).…”

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

et al. 2016

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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 ( NOS s) 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), 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 (N ω ‐hydroxy‐L‐arginine oxidation).

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The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

Cited by 20 publications

References 74 publications

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases

Exploring second coordination sphere effects in nitric oxide synthase

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

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