Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at

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Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-based Sensor YddV Jean-Christophe Lambry, Martin Stranava, Laura Lobato, Marketa Martinkova, Toru Shimizu, Ursula Liebl, and Marten H Vos J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.5b02517 • Publication Date (Web): 11 Dec 2015 Downloaded from http://pubs.acs.org on December 12, 2015

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The Journal of Physical Chemistry Letters

Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-based Sensor YddV

Jean-Christophe Lambry†, Martin Stranava§ Laura Lobato†, Marketa Martinkova§, Toru Shimizu§, Ursula Liebl†, & Marten H. Vos†*

†

LOB, Ecole Polytechnique, CNRS, INSERM, 91128 Palaiseau Cedex, France

§

Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova

(Albertov) 2030/8, Prague 4, Czech Republic

*corresponding author: [email protected]

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ABSTRACT An important question for the functioning of heme proteins is whether different ligands present within the protein moiety can readily exchange with heme-bound ligands. Studying the dynamics of the heme domain of the Escherichia coli sensor protein YddV upon dissociation of NO from the ferric heme by ultrafast spectroscopy, we demonstrate that, when the hydrophobic leucine residue in the distal heme pocket is mutated to glycine, in a substantial fraction of the protein water replaces NO as an internal ligand in as fast as ~4 ps. This process, which is near-barrierless and occurs orders of magnitude faster than the corresponding process in myoglobin, corresponds to a ligand swap of NO with a water molecule present in the heme pocket, as corroborated by molecular dynamics simulations. Our findings provide important new insight into ligand exchange in heme proteins that functionally interact with different external ligands.

TOC Graphics

Keywords: Molecular dynamics, heme proteins, ultrafast spectroscopy, simulation, nitric oxide

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Numerous heme proteins functionally exploit their ability to bind various external or internal molecules as axial ligands to the heme iron. These can be substrate, intermediate or a product in enzymatic catalysis, a sensing ligand in signal transduction or a stored ligand, as in the case of the oxygen storage and transport proteins myoglobin and hemoglobin. The heme-ligand bond can often be photodissociated and this feature is extensively used to spectroscopically follow ligand pathways through the protein, as well as ligand-heme rebinding. Many heme proteins, in particular sensor proteins1, actually function by exchanging ligands. In some systems, such as EcDos2, CooA3 or neuroglobin4, an internal ligand residue and an external gaseous ligand exchange. Here, whereas rebinding of the internal residue typically occurs on the time scale of a few picoseconds5-8, dissociation and migration out of the heme pocket of the gaseous ligand generally lead to at least ~7 orders slower binding of the internal ligand (>~100 µs)6,9-11, a process limited by the reorganization of the protein moiety required for repositioning the residue. The situation can be different in the case of an exchange between two different small exogenous ligands, where substantial protein rearrangement is not necessarily required. Whereas ligand exchange between heme-bound ligands and ligands in the solvent can be studied by stopped-flow and flash-flow types of spectroscopy, the question arises whether ligands present within the protein moiety can readily exchange with heme-bound ligands. Studying this issue experimentally is challenging, because of the requirement of macroscopic samples with a well-defined initial state, with one type of ligand bound to the heme and the other docked within the protein moiety. Yet, in myoglobin, ~10% of the protein has been reported to bind a water molecule after dissociation of NO from the ferric heme12. This process occurs on the timescale of hundreds of nanoseconds and is presumably limited by diffusion of water into the heme pocket after the latter has been emptied by NO. Until now, no protein system has been described where ligand exchange between two different molecules

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simultaneously present in the heme pocket can be time-resolved. Here, studying a genetically modified heme domain of a bacterial sensor protein, we demonstrate that the intrinsic replacement speed of NO by a water molecule as a heme ligand is much faster (a few picoseconds) when the water molecule is present in the heme pocket at the instant of NO dissociation. These results have important general implications for our understanding of the mechanism of functional exchange between different ligands in heme proteins. YddV from Escherichia coli is a diguanylate cyclase enzyme whose activity is regulated via a heme-containing globin sensor domain13,14. This sensor domain, of which the structure has only very recently been described15, shows sequence and structural similarity with that of HemAT from Bacillus subtilis16. YddV acts in tandem with the phosphodiesterase EcDos13,17, whose enzymatic activity itself is under control of a heme-containing PAS sensor domain, in regulating cyclic di-GMP homeostasis. YddV displays activity when its heme is in the ferric unliganded or ferrous oxy or carbonmonoxy forms and is inactive in the ferrous unliganded or NO-bound forms; it has been suggested that its main physiological role is oxygen sensing13,17. Site-directed mutagenesis studies have highlighted the crucial role of the distal heme pocket residues Tyr43 and Leu65 in the ligand affinity, stability of the oxycomplex, and heme pocket compressibility17-20. In particular, Leu65 was found to be important for blocking the access of water molecules into the heme pocket, thus slowing autoxidation of the oxycomplex18. Indeed, the Fe(III) complex of the YddV wild type protein was found to be five-coordinated, whereas mutation of Leu65 allowed formation of the sixcoordinated, H2O-bound complex. Here, we explore the implications of the difference in water accessibility in the context of the ferric NO complex, which has not been studied previously, of the YddV heme domain (termed YddV-heme). Fig. 1A depicts a model of this complex.

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Figure 1. A. WT ferric YddV-NO complex. The figure, highlighting key residues, corresponds to a snapshot at the end of the equilibration phase of the molecular dynamics simulation of the model, which is based on homology between YddV and HemAT. B. Ground state absorption spectra of the Fe(III) (solid) and Fe(III)-NO (dashed) forms of WT (red) and L65G (blue) YddV-heme. Inset: corresponding Fe(III) minus Fe(III)-NO difference spectra in the Soret region. These are used as model difference spectra for NO dissociation and replacement of NO by H2O as a ligand (Fe(III)-H2O minus Fe(III)-NO) respectively.

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Fig. 1B shows the absorption spectra of the Fe(III) forms of WT and L65G mutant YddV-heme before and after exposure to NO. For WT and the mutant, exposure to NO leads to very similar spectra (with maxima at 418, 534 and 568 nm) that are typical for the Fe(III)NO form21,22. As reported previously18, the spectra for the Fe(III) form in the absence of gaseous ligands are different for WT and L65G YddV-heme, in particular in the Soret band with maxima at 393 and 406 nm, reflecting the 5-coordinated and H2O-bound 6-coordinated ferric heme, respectively. The corresponding difference spectra (Fig. 1B, inset) can therefore be regarded as model spectra, expected for NO dissociation from the heme and of the replacement of NO by water as a heme ligand.

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Figure 2. Dynamics after dissociation of NO from WT YddV-heme. A Transient absorption spectra at selected delay times and (inset) kinetics at selected wavelengths, indicated by arrows in the spectra. Solid lines in the kinetics are from a global fit. B Solid lines: Evolution Associated Spectra obtained from global analysis of the data set in terms of a 4-exponential spectral evolution. The precursor spectra evolve in 7 ps (black), 90 ps (red), 650 ps (green) and > 4 ns (blue). Dashed line: normalized steadystate NO-dissociation spectrum (red curve in inset Fig. 1).

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Fig. 2A shows the spectral evolution of the WT Fe(III)-NO complex upon NO photodissociation. As with dissociation of other ferric heme-NO complexes23-26, a blue shift of the Soret band is observed (Fig. 2A), which strongly resembles the static unliganded minus NO liganded spectrum (Fig. 1B, red line in inset; Fig. 2B). Global analysis of the data in terms of a multiexponential model yielded a good fit with four exponentials (Fig. 2B). The fastest (~7-ps) component concerns the relaxation of a small red shift and is assigned to a heme thermal relaxation process that typically occurs on this timescale27. On this timescale, no evolution on the blue induced absorption side of the spectrum is observed, implying that no sizeable change in heme coordination occurs. The remaining components have very

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similar spectral shapes and can all be safely assigned to direct recombination of NO with the heme. Overall, multiphasic recombination of the dissociated NO with the 5-coordinated heme occurs for ~80% until the end of our 3.5 ns time window, on the tens of picoseconds to nanoseconds timescale. Time constants of 90 ps (~25% of the dissociated Fe(III)-NO complexes), 650 ps (~45%) and >4 ns (~30%) were found in the multiexponential model; the extent and rate of the longest component could not be well established. Nonexponential, multiphasic geminate Fe(III)-NO rebinding is often encountered in ferric NO complexes23,25,28 and presumably reflects static or dynamic heterogeneity in the configurations sampled by the dissociated NO. Our results indicate that a substantial fraction, but less than 20%, of the dissociated NO can escape the heme environment and migrate out of the protein. We note that as in nitrophorin23, in YddV the NO rebinding to ferric heme is substantially slower than to ferrous heme, where the dominant rebinding phase takes ~5 ps (data not shown). The initial spectrum observed upon dissociation of NO from the L65G mutant complex is very similar to that of WT (Figs. 2A, 3B), reflecting formation of the 5coordinated Fe(III) complex. However, in contrast to WT, the shape of the spectrum changes throughout the investigated picosecond and nanosecond timescale. The induced absorption band, initially peaking at ~388 nm, gradually shifts to the red, including on the timescale of a few picoseconds, where no spectral evolution occurs in WT. The final spectrum has a maximum at ~401 nm and is very similar in shape to the steady state H2O minus NO bound spectrum (Fig. 1B, blue line in inset; Figs. 3A, 3B). We conclude that in a substantial fraction of the NO-dissociated hemes, water binds at the vacant 6th coordination site and thus efficiently competes with NO. Quantitative analysis indicates that this fraction amounts to ~25% (Fig. 3B).

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Figure 3. Dynamics after dissociation of NO from L65G YddV-heme. A Transient absorption spectra at selected delay times and (inset) kinetics at selected wavelengths, indicated by arrows in the spectra. Solid lines in the kinetics are from a global fit. B Solid lines: Evolution Associated Spectra obtained from global analysis of the data set in terms of a 4-exponential spectral evolution. The precursor spectra evolve in 4.1 ps (black), 90 ps (red), 500 ps (green) and > 4 ns (blue). Dashed black line: normalized fitted initial spectrum in WT (black solid curve in Fig. 2B). Dashed blue line: normalized steady-state H2O minus NO spectrum shape of the fitted initial spectrum (blue curve in inset Fig. 1). The amplitude of this spectrum corresponds to 25% of the dissociated population, using the initial precursor spectrum for normalization with WT.

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The spectral evolution can be described with time constants of 4.1 ps, 90 ps, 500 ps and a long-lived component. These time constants are similar as in WT, yet the overall decay is substantially faster, mostly because the relative weight of the 90 ps component is higher. The 4.1 ps component includes both the spectral relaxation at the red side of the spectrum observed also in WT and the shift of the blue induced absorption band due to water binding (Fig. S1). These distinct processes take place on similar timescales and are not kinetically separated in our experiments. The 90 ps and 500 ps phases are virtually exclusively associated with rebinding of NO to the heme (Figs. S1, S2). We conclude that H2O binding occurs

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predominantly during the 4.1 ps phase. Overall, a picture of the L65G system emerges where in 25% of the excited complexes H2O replaces the photodissociated NO within a few ps. The remaining 75% of dissociated NO completely rebind in a slower, multiphasic way on the tens and hundreds of picoseconds timescale. Thus, NO and H2O do not appear to directly compete in binding to the heme, otherwise spectrally mixed phases would have been observed. Rather, we propose that in one fraction (fraction I, Scheme 1) a H2O molecule is present close to the heme that can efficiently coordinate to the heme, thus displacing the dissociated NO, whereas in the (larger) fraction II, no H2O molecule is suitably located in the heme pocket and consequently NO can rebind. The fact that, in contrast to WT, no significant escape from the heme pocket is observed in fraction II of the L65G system, suggests that here the water molecules actually constrain the NO near the heme.

Scheme I Schematic representation of NO and H2O rebinding to the heme iron of L65G YddV-heme.

We have also investigated the dynamics in other leucine 65 mutant proteins of YddV. L65M, L65Q, and L65T YddV-heme also form, at least partly, a Fe(III)-H2O, complex, as does L65G18. In these systems, the dynamics of the Fe(III)-NO complex were qualitatively similar as reported here for L65G. Unfortunately, the Fe(III)-NO complex of these systems undergoes quite fast auto-reduction, leading to substantial contributions of the Fe(II)NO 9



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complex to the dynamics, which complicates the analysis. Upon mutation of tyrosine 43, another important residue composing the heme distal site, the Fe(III) complex remains 5coordinated17, indicating that H2O access remains blocked. Consistently, in the Fe(III)-NO complexes of such mutants, we did not observe transient formation of a Fe(III)-H2O complex. To investigate the activation barrier for migration of the water molecule and binding to the heme upon NO dissociation, we determined the temperature dependence of the ~4 ps, water binding phase in the range 8-32 °C (Fig. S3). We found almost no change in the rate over this range; hence the process is near barrierless (Ea = 6 ± 3 kJ/mol). For comparison, the barrier observed for H2O migration to the heme pocket and heme-binding to NO-dissociated myoglobin is 42 ± 3 kJ/mol)12. The low activation barrier observed in the YddV-heme L65G system supports the notion that the H2O molecules that can bind to the heme are already close to the heme prior to NO dissociation and do not migrate into the heme pocket from the outside. In an attempt to grasp the molecular origin of the observed picosecond H2O binding in YddV-heme with replaced leucine 65, molecular dynamics simulations were performed of the NO ligand dissociation from ferric heme iron in WT and L65G YddV models. Note that such classical simulations do not take into account heme-ligand bond (re)formation. We found that after dissociation, on the tens of picoseconds timescale in both, WT and L65G, the NO ligand stays in the heme pocket at a 4 Å average distance from the heme iron. However, the proximity of H2O molecules was very different in both models. Firstly, Fig. S4 shows that the number of distal water molecules is much lower in WT (1-2 on average within 8 Å from the heme plane center) than in L65G trajectories (3-4 on average), in agreement with the earlier assessment that replacement of the hydrophobic residue leucine 65 opens the heme pocket to water molecules19. Interestingly, Fig. S4 also shows, particularly clear for WT, a decrease in distal waters on the tens of picoseconds timescale after Fe(III)-NO dissociation as a result of

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NO pushing away water molecules. Secondly, importantly, the distribution of the geometric parameters of the nearest distal water molecule during the simulations differs markedly between the two models on essential points (Fig. 4). In WT, almost all H2O molecules are at 6-9 Å of the heme plane center, and never within 3.2 Å. The L65G model shows a different pattern: here two roughly equally populated regions (3–6 Å and 7–8.5 Å) are obvious and water molecules do approach the heme plane center much more closely, to distances below 2 Å (in the aquamet complex of myoglobin the Fe-O bond length is ~2.0-2.3 Å29), including in orientations favorable for iron ligation (H-atoms pointing away from the heme). As an example, a movie showing one temporal swap of NO and water near the heme is shown in the Supporting Information. Altogether, these simulations indicate that the opening of the heme pocket to water, induced by replacing leucine 65 with glycine, allows transient population of heme-water configuration from which bond formation on the picosecond timescale is far more favorable. This is different from WT YddV-heme, where NO dissociation does not lead to configurations favorable for water binding. This observation is in general agreement with the recently proposed role of Leu65 in controlling the CO binding dynamics to the heme30. Figure 4. Configuration of the nearest distal water molecule during 40-ps simulations after NO dissociation. The minimal distance between a water molecule, oxygen atom and the center of the heme plane and the corresponding angle between the heme plane normal and the water plane symmetry axis is plotted for each point taken at 10-fs intervals from 7 independent trajectories. Chain A points are in black and chain B points in red.

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In conclusion, our results provide the first direct evidence of exchange between chemically different ligands at an intra-protein binding site occurring on the timescale of a few picoseconds. Here, the Fe(III)-NO bond is broken by photolysis, mimicking the native thermal dissociation process. Water, which acts both as solvent and ligand for the YddV L65G protein, has a lower affinity than NO, but can transiently bind to the heme iron if it is located at the heme distal side at the time of the bond breaking event. The dynamics of this exchange are on a similar time scale as the fastest phases of geminate rebinding observed in ferric and ferrous heme proteins27. Whereas Fe(III)-water geminate rebinding cannot be directly studied (the Fe(III)-H2O bond is not photolabile31,32), this comparison indicates that water binding is not much hindered by the presence of the dissociated ligand, an assessment corroborated by our simulations pointing at formation of Fe(III)-water configurations favorable for bond formation within picoseconds after NO dissociation. Instead, the second ligand in the heme pocket effectively hinders the initially bound ligand to rebind. This hindrance favors exiting of the initially bound ligand from the heme pocket compared to the situation where no other ligand is present. In general terms, this fast intra-protein ligand replacement process implies that exchange between ligands may occur faster than deduced from dissociation and bimolecular binding rates of the individual ligands based on steadystate affinity determinations and mixing techniques. Whereas the physiological relevance of the here studied YddV L65G mutant system is probably limited, this assessment may have implications for heme proteins that functionally interact with different external ligands, including many heme-based sensor proteins. Importantly, upon fast strong change in ligand environment, the exchange rate of the initially bound ligand may be limited by the thermal dissociation rate of the heme-ligand bond, rather than by the overall koff of the initially bound ligand, which also depends on the escape probability of the heme-dissociated ligand from the heme pocket. In future work, we will investigate whether such fast ligand exchange processes

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also occur in non-globin heme-based sensor domains, including PAS-domains (cf. Ref. 14), as well as in ferrous heme proteins.

Experimental and computational methods Proteins were expressed and purified as described17,18 and suspended at a concentration of ~50 µM in 20 mM Tris/HCl buffer, pH 8.0. Experiments were performed in 1-mm path length optical cells sealed with a gastight stopper. Oxygen was removed by several cycles of vacuum pumping and exposure to pure argon gas. Subsequently the gas phase was replaced by a mixture of 10% NO and 90% argon. The samples were left to equilibrate until formation of the Fe(III)-NO complex was completed as monitored by the steady-state absorption spectrum. Multicolor femtosecond absorption experiments were performed on a 500 Hz repetition rate setup as described33, with a pump pulse centered at 570 nm and a broad band continuum probe pulse extending down to ~350 nm generated in a continuously translated CaF2 window. Data were globally analyzed in terms of multiexponential decay using the Glotaran package34. A model of the YddV heme domain (residues 1-153) dimer was built based on the homology with the HemAT sensor domain from B. subtilis, and molecular dynamics simulations of the ferric NO complex of the WT and L65G mutant were performed before and after dissociation of the Fe(III)-NO bond as described in the Supporting Information section.

Acknowledgments. We are grateful to Dr. Kenichi Kitanishi for valuable discussions during the initial stage of this work. This work was supported in part by Agence National de la Recherche Grant ANR 09-PIRI-0019 (to MH.V. and U.L) and Grants-in-Aid from Grant Agency of the Czech Republic (grant 15-19883S) and Grant Agency of Charles University in

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Prague (grant 756214) and UNCE 204025/2012 (to M.M.). L.L. was recipient of a two-year fellowship from “La Caixa”, Spain.

Supporting Information Available: One file containing four supporting figures and MD simulation methods including one more figure. One file containing a movie with part of a trajectory after Fe(III)-NO dissociation in the L65G model. This material is available free of charge via the Internet at http://pubs.acs.org.

References

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TOC Graphics

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Figure 1.

0.8

406

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Absorbance

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Figure 2.

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Scheme I

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Figure 4.

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Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at

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