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Kinetic Control of O Reactivity in H-NOX Domains Yuhan Sun, Abdelkrim Benabbas, Weiqiao Zeng, Sandhya Muralidharan, Elizabeth M Boon, and Paul M. Champion J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b03348 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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The Journal of Physical Chemistry

Kinetic Control of O2 Reactivity in H-NOX Domains

Yuhan Sun1, Abdelkrim Benabbas1, Weiqiao Zeng1‡, Sandhya Muralidharan2, Elizabeth M. Boon2 and Paul M. Champion1* 1

Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, MA 02115

2

Department of Chemistry and the Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY 11794

Corresponding Author *Address correspondence to Paul M. Champion, Tel.: 617-373-5705; E-mail: [email protected]

‡

Present address: Department of Chemistry and Biochemistry, Utah State University,

Logan, UT 84322

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Abstract Transient absorption, resonance Raman, and vibrational coherence spectroscopies are used to investigate the mechanisms of NO and O2 binding to WT Tt H-NOX and its P115A mutant. Vibrational coherence spectra of the oxy-complexes provide clear evidence for the enhancement of an iron-histidine mode near 217 cm-1 following photoexcitation, which indicates that O2 can be dissociated in these proteins. However, the quantum yield of O2 photolysis is low, particularly in the wild type (≲3%). Geminate recombination of O2 and NO in both of these proteins is very fast (~1.4x1011 s-1) and highly efficient. We show that the distal heme pocket of the H-NOX system forms an efficient trap that limits the O2 off-rate and determines the overall affinity. The distal pocket hydrogen bond, which appears to be stronger in the P115A mutant, may help retard the O2 ligand from escaping into the solvent following either photoinduced or thermal dissociation. This, along with a strengthening of the Fe-O2 bond that is correlated with the significant heme ruffing and saddling distortions, explains the unusually high O2 affinity of WT Tt H-NOX and the even higher affinity found in the P115A mutant.


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Introduction The heme nitric-oxide/oxygen binding domain (H-NOX) occurs in a newly discovered family of heme-based sensor proteins, widely existing in both aerobic and anaerobic bacteria. In eukaryotes, the H-NOX domain is found in soluble guanylate cyclase (sGC), which is the paradigm of mammalian NO sensors.1 In prokaryotes, the HNOX proteins appear to fall into one of two classes. One type is a stand-alone protein most often found within a predicted operon along with a histidine kinase or, less frequently, with a diguanylate cyclase domain. The other class is fused to methylaccepting chemotaxis domains in the same open reading frame.2-4 The H-NOX domains from the majority of eukaryotes and facultative aerobic prokaryotes do not bind O2, but they do bind NO and, upon losing the proximal histidine ligand, form a five-coordinate (5C) complex that is similar to sGC-NO. On the other hand, H-NOX proteins from obligate anaerobic prokaryotes, including Tt H-NOX, bind both NO and O2 and form stable six-coordinate (6C) complexes.5-7 Homology to sGC as well as genomic placement suggests that H-NOX domains in prokaryotes are likely to serve as sensors for gases such as O2 and NO. Recent results that probe the So H-NOX protein from Shewanella oneidensis4, as well as other facultative aerobes,8-9 are consistent with this hypothesis. Understanding heme-ligand interaction and the mechanisms of ligand recognition in this important class of heme proteins is a key to understanding their biological functions.7, 1012

The crystal structure of O2 bound Tt H-NOX has been recently reported and an important structural feature is that it has the most highly distorted heme observed to date.13 The out-of-plane heme distortions found in Tt H-NOX show large deviations from



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planarity. This distortion appears to be caused by van der Waals interactions in the heme cavity, with residue Pro115 making the largest contribution (Fig. 1). Pro115 is within van der Waals contact with pyrrole-D of the heme, causing the pyrrole to shift out of plane, generating a large kink in the heme propionate group. The mutation of P115 to Ala, drastically reduced the OOP distortions as expected.14 Using the normal coordinate structural decomposition (NSD) method, we found that the heme out-of-plane (OOP) distortions are mainly along the ruffling and saddling directions with amplitudes 3.8 and 3.7 amu1/2Å, respectively, whereas in the mutant these distortions are reduced to ~2 and ~ 1 amu1/2Å, respectively. This is shown graphically in Fig. 1 where it should be noted that the iron atom is included in the NSD analysis and mass-weighted normal modes are therefore utilized (e.g., for a carbon atom displacement of 1 Å in Cartesian coordinates, the mass-weighted coordinate displacement is 3.46 amu1/2Å). Heme OOP distortions are often conserved for those proteins that belong to a given functional class.15-17 For example, doming is typically observed in oxygen storage or transport proteins like hemoglobin18-19 and myoglobin.20 Ruffling is the dominant OOP deformation found in c-type cytochromes15-17, 21 and nitrophorins22-25, which are involved in electron and NO transport, respectively. Although oxygen is a ubiquitous heme ligand in many biological systems, its interaction with heme proteins is only rarely investigated using non-linear spectroscopic or kinetic methods.26-28 This is due to the inherent instability of the oxy complexes and the propensity for photooxidation upon exposure to laser radiation. Consequently, the mechanism by which O2 interacts with the heme, in the process of carrying out key biological functions such as sensing and catalysis, still remains somewhat elusive. The


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differences in heme distortion found for the O2 complexes of Tt H-NOX and its P115A mutant provide a unique opportunity to study how heme distortion might be related to the O2 binding function. Vibrational coherence spectroscopy (VCS) has the ability to probe the vibrational modes of heme proteins below 200 cm-1 in an aqueous environment. We have previously investigated the low-frequency modes of a variety of heme proteins, using Soret band excitation VCS.20, 29-35 Unlike higher frequency Raman modes (>200 cm−1), the protein surroundings can more easily distort the heme away from equilibrium along the lowfrequency out-of-plane modes because of their weaker force constants. These modes are observed in VCS due to the symmetry-breaking nature of the non-planar heme distortions.32 In addition, these modes take on potential functional significance because of the thermal population of their vibrationally excited states. The low-frequency vibrational coherence spectra offer a unique window into how the surrounding protein environment can alter these potentially important and thermally active heme modes. In this study, we use transient absorption, resonance Raman (RR) and VCS spectroscopies to systematically investigate the photolysis and rebinding dynamics of O2 and NO in wild type (WT) Tt H-NOX and its P115A mutant. We find that the quantum yield for O2 photolysis in this protein is unusually small and suggest that specific heme OOP distortions (i.e., ruffling and/or saddling) contribute to the stabilization of the Fe-O2 bond of Tt H-NOX. The combination of an unusually strong Fe-O2 bond, along with a “trap” for the dissociated O2 in the distal heme pocket, ensures a rapid geminate recombination rate and a very high affinity for O2.



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Results and Discussion Absorption and Raman Spectra. Absorption spectra of ferrous Tt H-NOX and its P115A mutant, along with the spectra of their NO and O2 adducts, are shown in Fig. S1 of the supporting information (SI). Steady state difference spectra of the unligated protein minus the ligated protein, which are proportional to the difference of the wavelengthdependent extinction coefficients, are displayed in the insets of Fig. S1. Resonance Raman spectra, obtained with 413.1 nm excitation, are shown in Fig. 2 and are in good agreement with previous studies.28 The positions of heme marker bands (ν4, ν3, ν2, ν10) are given in Table S1 of SI and clearly indicate that the heme is a 5coordinate (5C) complex in the ferrous state, whereas the NO and O2 adducts form 6coordinate (6C) low spin species. In the ferrous samples, a strong Fe-His stretching mode is observed near 220 cm-1, which is associated with the 5C histidine bound heme configuration. As expected, this mode is not observed in the NO or O2 complexes. There are noticeable differences between the spectra of ferrous Tt H-NOX and its P115A mutant in the low frequency region (200-600 cm-1), which has sensitivity to the ruffling and saddling deformations. Similar changes are seen in the O2 and NO complexes. These differences are attributed to the P115A mutation, which reduces the large OOP heme distortions found in the wild type.28 Some differences between the ferrous WT and P115A, and their respective NO and O2 adducts, are also observed in the high frequency Raman spectra. The oxidation marker band, ν4, of the NO adduct undergoes a small shift from 1373 cm-1 (WT) to 1375 cm-1 (P115A), while the O2 adduct shows a slightly larger shift from 1373 cm-1 to 1377 cm-1. The decreased ν4 frequency in the more ruffled WT protein agrees with previous findings.36 There are also clear differences in the ν3, ν2, and


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ν10 modes when the O2 complexes of the WT and mutant are compared (see SI Table S1). Finally, a weak feature associated with the Fe-O2 moiety has been assigned at 569 cm-1 and 567 cm-1 in the WT and the mutant respectively.28 This feature is not a pure Fe-O2 stretching mode because the hydrogen bonded distal oxygen atom motion is very small in comparison to the stretching motion between the proximal oxygen and the iron atom to which it is bound37. As such, this mode is more formally a Fe-O stretch with a significant admixture of Fe-O2 bending37. The presence of a hydrogen bond, regardless of its strength, evidently minimizes the distal oxygen atom motion and makes the “Fe-O2” mode less sensitive to the hydrogen bonding strength than might otherwise be expected. VCS Spectra. The VCS measurements of ferrous Tt H-NOX, the P115A mutant, and their NO complexes are shown in Fig. 3, while the data for the O2 complexes are displayed in Fig. 4. The VCS spectra of the unligated samples agree well with the corresponding Raman spectra in the region of overlap (~200-400 cm-1). When the WT and the P115A mutant samples are compared, they show a 6 - 9 cm-1 difference in the position of the strong low frequency mode found in the region ~30-50 cm-1. A downshift of this mode in the WT protein, where the heme is more ruffled, is seen in the ferrous and NO bound forms and this is consistent with prior studies that reveal anharmonic mode softening as the ruffling distortion is increased.32, 38 In contrast, the O2 bound complex shows apparently anomalous behavior, where a downshift of this mode is observed in the less ruffled P115A, which has a mutation close to the proximal His102 that relieves stress on the heme (Fig.1). In 5C ferrous heme systems, the iron is equilibrated out of the heme plane, breaking the D4h symmetry and allowing out-of-plane Raman activity so that the



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dominant low-frequency mode (~40 cm-1) is correlated with doming motion.35, 39 On the other hand, for systems where the major heme distortion is ruffling or saddling, like cytochrome c38, nitrophorin 432, and 6C H-NOX, the strongly allowed low-frequency mode(s) should involve motions that track with the square of the NSD distortion amplitudes.32 Thus, based on the strongly ruffled and saddled oxygen complex where structures are available (Fig. 1), we assign the main low frequency mode observed in the H-NOX protein to motions that have a large ruffling and/or saddling component. Although x-ray structures for the ferrous and NO bound Tt H-NOX protein are not available, the minimal structural changes observed upon NO binding to the So H-NOX MnII-substituted system40, suggests that the protein induced heme distortions do not change dramatically in the NO complex. The broader linewidths seen in the 5C ferrous VCS spectra, compared to the 6C ligand bound states in Figs. 3 and 4, probably arise from a combination of spectral congestion involving increased doming activity as well as a more rapid decoherence of the coherently excited heme vibrations. When compared to 6C heme complexes, disorder in the 5C heme is generally larger and leads to both inhomogeneous broadening of the optical spectra41 and kinetic heterogeneity in CO rebinding.42 Structural inhomogeneity within the 5C ensemble will lead to a distribution of out-of-plane mode frequencies and a more rapid decay of the coherence signals. The six coordinate NO and O2 adducts have VCS spectra that are potentially complicated by photolysis. However, for the low photo-dissociation yield O2 complexes, the spectra are comprised of mostly the 6C form (vide infra). Thus, based on the NSD analysis, the modes at 51 cm-1 (WT-O2) and 42 cm-1 (P115A-O2) are expected to be


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primarily associated with ruffling32, 34 although and admixture of saddling could also be present. As observed for the ferrous and NO bound H-NOX samples, low-frequency ruffling/saddling heme modes are generally anharmonic and “soften” (i.e., shift to lower frequency) as the magnitude of the distortion increases.38 However, in the case of the O2 complex there is an unusual upshift observed for the more distorted WT compared to the less distorted mutant protein. This suggests that additional factors may be involved in controlling the frequency of the ruffling and saddling modes when oxygen is bound to the heme. One possibility involves the H-bond between O2 and the Tyr140 which stabilizes the excess electron density on the bound O2. A particularly strong H-bonding interaction should increase the electron density transferred from the heme orbitals to the bound O2 in the spincoupled bonding arrangement. Loss of electron density in the heme orbitals would be expected to soften the force constants associated with out-of-plane heme motions. Because there appears to be a significantly stronger H-bond between Tyr140 and the bound O2 in the mutant (i.e., ~2.3Å distance14 between the H-bonded oxygen atoms of P115A compared to the ~2.7Å distance13 in the WT), the out-of-plane heme force constants of P115A-O2 would be reduced, and help explain the unusual downshift of its low frequency modes relative to the more distorted WT. NO Rebinding Kinetics. Kinetic measurements of the different samples, shown in Fig. 5, were performed using the VCS setup by taking data over a longer time scale (~200 ps). Each curve in Fig. 5 is typically an average of 3-4 runs. The kinetic traces tracking change in transmission (∆T/T) were fit with a simple multi-exponential decay function that can be written as

∆

= ∑ exp−/ + . Coherence coupling signals,



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which are due to the overlap between pump and probe pulses near time zero, were trimmed before fitting the data. The results of the different fits are summarized in Table S2 of the SI. The value of n is chosen to be the minimum value necessary to fit the data with an adjusted R-square higher than 0.99. For the unligated ferrous samples, both the WT and P115A mutant have a similar ~4 ps time constant with positively signed ΔT that dominates the decay. This rate has been assigned to vibrational cooling of the transiently heated heme, following photon absorption.26 The kinetic traces of the NO complexes in both proteins show a fast decay component (0.22 ps) and a subsequent slow rise back to ∆T=0 with ~7 ps time constant. The fast rate may be associated with an electronic relaxation component. The slower time constant, however, is assigned to geminate NO rebinding. Indeed, the VCS spectra of NO complexes (Fig. 3) show the appearance of a Fe-His mode around 225 cm-1, indicating that NO dissociates upon photo-excitation. The NO rebinding rates are 1.34×1011 s-1 and 1.60×1011 s-1 for the WT and the mutant, respectively. These values are consistent with the rates of NO rebinding to other ferrous heme proteins.25, 43-44 More importantly, they agree very well with the rate (1.34×1011 s-1) of NO rebinding to the homologue protein sGC.44-45 These results show that the geminate NO rebinding rate within the H-NOX family is independent of the extent of heme distortion or its coordination state (5C or 6C). The invariance in the NO kinetics is consistent with the “harpoon” model of NO geminate rebinding and with the absence of temperature dependence in this rate.26, 43 O2 Photolysis Quantum Yield. The VCS spectra of the O2 complexes in Figs. 4a and 4b show the activation of a transient photoproduct. A Fe-His mode is detected at 214 cm-1 in


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the WT, reflecting a somewhat weakened Fe-His bond (probably due to a larger Fe-His tilt than found in the NO complex), and at 218 cm-1 in the mutant, which reflects a less distorted heme with a slightly stronger and less-tilted14 Fe-His bond. The observation of the Fe-His mode in Fig. 4 is direct evidence that O2 photo-dissociates in both proteins. The Fe-His mode is observable, even though the population of the photolyzed species is small, because the coherence amplitude scales with the magnitude of the equilibrium shift. This shift can be quite large for modes associated with moving the heme structure from the 6C to the 5C equilibrium state. The kinetic traces of the O2 complexes probed at 435 nm (Fig. 5) can be fitted with 3 exponentials and an offset (Table S2 of SI). The extracted time constants are 1.7 ps, 7.4 ps, and 64 ps for the WT-O2 protein, which is shown on a semi-log plot in Fig. 5 in order to better visualize the three kinetic phases. The time constants for the P115A-O2 mutant are 0.6 ps, 6.6 ps, and 60 ps (the latter phase is seen more clearly on a linear scale in the insert). The shortest and longest time constants observed in the WT and mutant O2 kinetics cannot be assigned to O2 rebinding because the amplitudes have the wrong sign (Table S2 of SI). Indeed, the steady state difference spectra (ferrous minus O2 complex) shown in the inset of Fig. S1 of the SI indicate that the change in the absorbance ∆! due to ligand photolysis should have a positive sign when probed at 435 nm. Correspondingly, the ∆"/" signal, which describes the decay of the photolyzed population, should have a negative sign. Hence, we attribute the time constants of 7.4 ps and 6.6 ps to O2 rebinding in Tt H-NOX and its P115A mutant, respectively. These time constants agree quite well with the fastest time constant (6 ps) found for O2 rebinding in Mb.26



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Sub-picosecond time resolved resonance Raman spectroscopy46 has been used to investigate O2 rebinding kinetics in FixL. The absence of Fe-His mode in the early time Raman spectra was attributed to a very fast (within ~ 100 fs) O2 rebinding. Although the time resolution of our experimental setup is (~70 fs) we did not observe any subpicosecond O2 rebinding component with negative ∆T in either Tt H-NOX or its mutant. The slowest observed pump-probe optical response for the O2 samples has a small positively signed ∆T amplitude and a time constant ~60 ps, suggesting that it arises from a structural relaxation of the heme surroundings. As a result of the tight packing of the heme pocket of H-NOX, some of the photon energy deposited in the heme of the unphotolyzed O2 complex can be transferred to nearby residues via the van der Waals interactions between the heme and P115 (somewhat less so through the A115 contact, which also has a smaller heme distortion correlated with the reduced amplitude of the 60 ps optical response of the P115A mutant). If the surrounding residues of the unphotolyzed 6C population are structurally altered by thermal energy from an absorbed photon, it would lead to perturbations of the optical absorption. We suggest that the structure relaxes back to equilibrium with the ~60 ps time constant, but with larger amplitude for the WT protein.

This is consistent with both the smaller photolysis

quantum yield in WT (i.e., a larger vibrationally hot and unphotolyzed 6C population) as well as its larger heme distortion arising from strong contact with P115. For the same reason, the positively signed small amplitude slow component (20- 30 ps, Table S2 of SI) detected in the unligated samples can also be attributed to a thermally induced protein structural relaxation process. On the other hand, the NO complex does not display a slower structural relaxation response. We attribute this to the much larger quantum yield


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for ligand photolysis, which evidently dissipates a large fraction of the photon energy in breaking the Fe-NO bond, rather than in structural perturbation and/or “heating” of the heme surroundings. The photolysis quantum yield (#$% ) of Tt H-NOX-O2 and its mutant can be estimated using the procedure outlined in the SI. Based on the similarity of the relative amplitudes of the two phases observed in the NO kinetics, we assume that the photolysis quantum yield of NO is similar for the WT and P115A mutant and take its value to be roughly #&$ ∼50%, as found for Mb26, or possibly larger. Using #&$ ∼50% as a reference point, we can estimate that the O2 photolysis quantum yield is 1.5% and 7.5% in WT Tt H-NOX and its P115A mutant, respectively. These values are much lower than the O2 photolysis quantum yield of Mb.26 More generally, if we consider that NO dissociates from both proteins with unity quantum yield, we can find the upper limits for the O2 photolysis quantum yield for WT (#$% < 3%) and the P115A mutant (#$% < 15%). Data that confirm this analysis can be found in Fig. 4b, where it can be seen that the amplitude of the Fe-His oscillation is ~5-6 times larger in the mutant compared to the WT, although the WT coherence is longer lived. This is consistent with an increase of the O2 photolysis quantum yield in P115A by a factor of ∼5 compared to the WT. This also suggests that the Fe-O2 bonding arrangement is stronger in the WT than in the mutant. Kinetics Factors Controlling O2 Affinity. It is well established that the distal pocket Hbond network, especially the H-bond between O2 and Tyr-140, plays a major role in stabilizing the oxy complex in H-NOX proteins.4, 10-11, 14, 47-48 However, this factor alone cannot explain why the Fe-O2 bond in the WT Tt H-NOX is more robust against photolysis in comparison to the P115A mutant. In fact, the O2-Tyr140 H-bond in the WT



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is longer (2.7 Å) than what is found in the mutant (2.3 Å), suggesting that the WT actually has a weaker H-bonding arrangement. Thus, it appears that an additional factor must play a role in the stabilization of the WT Fe-O2 bond against photolysis. We examined the effects the Fe-O-O angle and the Fe-His tilt and found that the Fe-O-O angle is the same (~120o) in both proteins, while the extent of the Fe-His tilt favors a stronger 14, rather than a weaker, Fe-O2 bond in the mutant compared to the WT. Thus, a remaining factor, which might explain the robustness of Fe-O2 bond against photo-dissociation in the WT protein compared to the P115A mutant, is the large difference in the heme ruffling and saddling distortion. As the dxz and dyz orbitals of the iron atom become more localized in the strongly ruffled heme system38, they will be available to strengthen the π-bonding antiferromagnetic exchange interaction that is very important in establishing the Fe-O2 bond in heme proteins.49 Although, the results indicate that WT Tt H-NOX is more robust against photolysis than its P115A mutant, recent studies have shown that the WT has a lower affinity for O2 (KD = 90 nM) compared to the P115A mutant (KD = 21 nM)14, which might initially appear contradictory. Because the O2 association rates of the WT and mutant are nearly the same, the difference in affinity must be primarily due to the difference in the thermal dissociation rates (koff = 1.22 s-1 and 0.22 s-1 for the WT and mutant, respectively).14 The apparent contradiction can then be understood if we consider the results by using the three-state model for ligand binding and dissociation in a protein25,

koff =

50

(see SI for more details). In this model, the off-rate is approximated as

k AB kout k ≈ k AB out when the geminate rebinding rate, k BA is large (≫ kout ) as kBA + kout kBA


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found for H-NOX. The thermal bond breaking rate is given by k AB , while kout is the ligand escape rate from the distal heme pocket into solution. It is the rate constant k AB , rather than koff , that should be correlated with the strength the Fe-O2 bond. As mentioned above, the on-rate is effectively independent of the mutation14, so the off-rate controls the overall O2 affinity of Tt H-NOX and its mutant. The off-rate depends upon both the Fe-O2 bond strength and the potential for trapping the O2 near the heme iron. Trapping in the distal pocket will reduce /012 and the fraction of photolyzed molecules, 34 = /012 //56 + /012 , that escape into the solvent. The measured kinetics allow us to extract these fundamental rates and the detailed expressions for doing so are given in the SI. The final results are presented in Table 1, using the measured parameters that are listed in Table S2 of SI. As can be seen from Table 1 the fraction of the dissociated O2 molecules, 34 ≈ /012 //56 , that escape into the solvent is very small in both proteins, but it is at least 10 fold smaller in the mutant when compared to the WT. Table 1 also shows that the thermal bond breaking rate /65 is about a factor of 3 smaller for the WT Tt H-NOX, when compared to the P115A mutant, which indicates that the Fe-O2 bond is indeed stronger in the WT protein. Thus, the values found for /65 are consistent with the relative quantum yields for O2 photolysis, where the Fe-O2 bond in the WT Tt H-NOX is found to be more robust against photolysis compared to the mutant. We conclude that the higher affinity of P115A mutant for O2 is not due to a stronger Fe-O2 bond, rather it is due to trapping in the distal pocket, which prevents the O2 ligand from rapidly moving to the solvent. This trapping effect is what significantly lowers the escape rate (/012 ) in the P115A mutant.



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One possibility is that the mutant possesses a more closed and tightly packed distal pocket than the WT. This correlates very well with the stronger (i.e., significantly shorter) H-bond that is observed between O2 and Tyr-140 in the x-ray crystal structure of the mutant

13

. A stronger H-bond is also suggested by the anomalous downshift of the

low frequency heme modes of P115A-O2 compared to the more distorted WT-O2. Both the more tightly packed distal pocket and the H-bond, if it retards the escape dynamics following Fe-O2 bond breaking, would decrease /012 for the P115A mutant compared to the WT. The Fe-O2 bond strength can be inferred from either the thermal bond breaking rate or the photolysis quantum yield. Because the Fe-O2 bond strength is larger in the WT by both of these measures (Table 1), we suggest that the larger heme distortion present in WT Tt H-NOX plays an important role in controlling the strength of Fe-O2 bond. As shown elsewhere38, 51, ruffling and saddling distortions affect the iron orbitals that interact with the π-electrons of the porphyrin and localizes them on the iron atom. When they become localized, the Fe electrons are able to interact more strongly with the bound O2 via the exchange coupling that characterizes this unique bond in heme proteins.49 Thus, we attribute the stronger Fe-O2 bond in the WT protein, compared to the P115A mutant, to the larger heme ruffling/saddling distortion generated by the surrounding protein. Summary. We investigated the dynamics of O2 and NO binding to Tt H-NOX and its P115A mutant, which revealed the relevant kinetic factors that control the affinity of HNOX domains for O2. We found that O2 photodissociates in WT Tt H-NOX, but the photolysis quantum yield is quite low (≲3%) compared to the P115A mutant (≲15%). Both NO and O2 rebind to Tt H-NOX very rapidly ( k BA ~1.4x1011 s-1), indicating that the


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barrier for geminate ligand rebinding is very low. The O2 affinity in Tt H-NOX is controlled by a combination of ligand trapping, the strength of the H-bond between O2 and Tyr 140, and the strength of the Fe-O2 bond. A strong H-bond in the O2 complex of the P115A mutant is consistent with the anomalous downshift of its low frequency modes, even though it has a smaller heme distortion than the WT. The relative thermal stability of the Fe-O2 bond in the WT protein is increased compared to the P115A mutant and this is consistent with the relative photolysis quantum yields. This suggests that the larger heme distortions (i.e., ruffling and/or saddling) in the WT protein leads to a localization of electron density in the iron dxz, dyz orbitals that can strengthen the π-bonding exchange coupling between the bound oxygen and the iron. Finally, we note that a slow structural relaxation response is observed in both the low quantum yield O2 complex and the unligated ferrous protein, but not in the much higher quantum yield NO complex. This suggests that in the NO system, photon energy is dissipated by the bond breaking process, while in the other systems the photon energy is dissipated by structurally perturbing the surroundings. Materials and Methods Sample Preparation. Tt H-NOX and its P115A mutant were prepared according to previously published methods.3, 28 Samples are freshly prepared in 50 mM HEPES buffer with 50 mM NaCl at pH 7.5 before the spectroscopy measurements. The ferrous state was prepared by adding an excess amount of sodium dithionite (20 fold) under argon. Ferrous NO-bound complexes were obtained by adding 10 fold excess of sodium nitrite to the ferrous H-NOX solution. The O2-bound complexes were obtained by passing the ferrous H-NOX solution through a size exclusion gel column (Sephadex G-25, Sigma)



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equilibrated with HEPES buffer in air. Absorption spectra were recorded (U-4100, Hitachi) after the preparation procedures. For VCS experiments the final concentration (~100 µM) of protein sample was adjusted to O.D. = 1±0.05 in a 1 mm optical path length quartz sample cell at the selected excitation wavelength. Absorption spectra were taken following the experiments to confirm the integrity of the samples. Optical Systems. Resonance Raman spectra were obtained using a standard 90⁰ light collecting geometry and a single grating monochromator (Acton SP2500i with 1800 g/mm UV holographic grating, Princeton Instruments). Details of the setup have been described elsewhere.32 Samples were placed in a quartz cell (Precision Cells, Inc.) spinning at 6000 rpm and excited with ~10 mW of the 413.1 nm line from a krypton laser (Innova 300, Coherent). The VCS system has been described in detail elsewhere.31, 39 The time resolution is 70 fs and the open band scheme31-32, 34, 52 was used with a Si photodiode collecting the entire spectral bandwidth of the probe pulse. The LPSVD data analysis method 34 was used to extract oscillatory component of the VCS signal.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Table S1 shows the absorption spectra of ferrous WT Tt H-NOX and its P115A mutant, along with the spectra of the NO and O2 complexes. Table S1 shows Raman bands of ferrous Tt H-NOX and its derivatives. Table S2 shows the fitting parameters of the kinetic data in Figure 5. Supporting text includes a description of the estimation of the O2 photolysis quantum yield in Tt H-NOX and the kinetic analysis of O2 rebinding using the 3-state model.


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Acknowledgements This work is supported by grants from the NSF CHE-1243948 (PMC), CHE-0910771 (EMB), and ONR N00014-10-1-0099 (EMB).

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Figure Captions Figure 1. Distal pocket architecture in the oxy complexes of (a) wildtype (WT) Tt HNOX (PDB 1U55), and (b) P115A Tt H-NOX (PDB 3EEE). The amino acids that surround the heme are shown in colored sticks. The hydrogen bond distance between the carboxyl oxygen of the Tyr side chain and the O2 ligand is shown as a green dashed line, which is 2.7 Å for the WT and 2.3 Å for the mutant. Panels (c) and (d) show the NSD analysis of the heme OOP distortion for the two oxygen bound complexes. Figure 2. Resonance Raman spectra of ferrous wild-type and P115A Tt H-NOX, and their NO and O2 complexes. The laser excitation wavelength is 413.1 nm with a power of 10 mW. All spectra were recorded with the sample spinning at 6000 rpm. Figure 3. VCS oscillations and LPSVD generated power spectrum amplitudes. The oscillatory signals associated with the strongest low frequency mode for each sample are shown in blue. The excitation wavelengths are: 425 nm for ferrous WT Tt H-NOX and P115A, and 427 nm for the NO complexes. Figure 4. (a) The VCS signals and the LPSVD generated power spectrum amplitudes for WT and P115A Tt H-NOX O2 adducts using an excitation wavelength of 423 nm. The oscillatory signals associated with the strongest low frequency mode for each sample are shown in blue. (b) Comparison of the oscillatory behavior of the Fe-His mode in the oxy complexes of the WT and P115A mutant. The data are obtained by keeping all optical conditions fixed and comparing samples with the same absorbance, verifying that the relative signals are properly normalized. The smaller signal for WT reflects its smaller


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photolysis quantum yield. Note that dephasing (loss of coherence) of the Fe-His mode in the mutant sample takes place on a slightly faster timescale. Figure 5. The optical response of the ferrous WT and P115A Tt H-NOX samples are compared to the kinetic signals from the NO and O2 complexes. The excitation wavelengths are labeled in each panel. The exponential fits to the data, starting at 0.5 ps, are shown as red solid lines. The fitting results are summarized in Table S2 of SI. The WT O2 complex is plotted on a logarithmic scale in order to better visualize the small amplitude components at 7 ps and 60 ps. The O2 complex of the P115A mutant depicts the small amplitude 60 ps kinetic phase using an expanded insert.

Table 1. Measured and derived rates for O2 kinetics. Measured

WT

P115A

Derived

WT

P115A

Ig

0.973

0.998

kBA (1011 s-1)

1.31

1.517

kg (1011 s-1)

1.35

1.52

kout (109 s-1)

3.6

0.3

Is

0.027

0.0017

kAB (s-1)

45

129

koff (a) (s-1)

1.22

0.22

YO2 (b)(%)

≲3

≲15

(a) From Olea et al. (b) Upper limit quantum yield assumes 100% NO photolysis as a calibration standard (see text and SI). 14



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Figures Figure 1.


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Figure 3

Figure 4.


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Figure 5.



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Figure 1 254x190mm (300 x 300 DPI)

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