PRIFYSGOL BANGOR / BANGOR UNIVERSITY Engineering Proximal vs Distal Heme-NO Coordination via Dinitrosyl Dynamics Kekilli, Demet; Peterson, Christine A.; Pixton, David A.; Ghafoor, Dlzar D.; Abdullah, Gaylany H.; Dworkowski, Florian S. N.; Wilson, Michael T.; Heyes, Derren J.; Hardman, Samantha J. O. ; Murphy, Loretta M.; Strange, Richard W.; Scrutton, Nigel S.; Andrew, Colin R.; Hough, Michael A. Chemical Science DOI: 10.1039/C6SC04190F Published: 01/01/2017 Peer reviewed version Cyswllt i'r cyhoeddiad / Link to publication Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA): Kekilli, D., Peterson, C. A., Pixton, D. A., Ghafoor, D. D., Abdullah, G. H., Dworkowski, F. S. N., ... Hough, M. A. (2017). Engineering Proximal vs Distal Heme-NO Coordination via Dinitrosyl Dynamics: Implications for NO Sensor Design. Chemical Science, 2017(8), 1986-1994. https://doi.org/10.1039/C6SC04190F Hawliau Cyffredinol / General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. 14. Jun. 2020
Transcript
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Engineering Proximal vs Distal Heme-NO Coordination via DinitrosylDynamicsKekilli, Demet; Peterson, Christine A.; Pixton, David A.; Ghafoor, Dlzar D.;Abdullah, Gaylany H.; Dworkowski, Florian S. N.; Wilson, Michael T.; Heyes,Derren J.; Hardman, Samantha J. O. ; Murphy, Loretta M.; Strange, Richard W.;Scrutton, Nigel S.; Andrew, Colin R.; Hough, Michael A.Chemical Science
DOI:10.1039/C6SC04190F
Published: 01/01/2017
Peer reviewed version
Cyswllt i'r cyhoeddiad / Link to publication
Dyfyniad o'r fersiwn a gyhoeddwyd / Citation for published version (APA):Kekilli, D., Peterson, C. A., Pixton, D. A., Ghafoor, D. D., Abdullah, G. H., Dworkowski, F. S. N.,... Hough, M. A. (2017). Engineering Proximal vs Distal Heme-NO Coordination via DinitrosylDynamics: Implications for NO Sensor Design. Chemical Science, 2017(8), 1986-1994.https://doi.org/10.1039/C6SC04190F
Hawliau Cyffredinol / General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/orother copyright owners and it is a condition of accessing publications that users recognise and abide by the legalrequirements associated with these rights.
• Users may download and print one copy of any publication from the public portal for the purpose of privatestudy or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ?
Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access tothe work immediately and investigate your claim.
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ChemicalScience
ISSN 2041-6539
Volume 7 Number 1 January 2016 Pages 1–812
EDGE ARTICLEFrancesco Ricci et al.Electronic control of DNA-based nanoswitches and nanodevices
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Petersen, D. Pixton, D. Gahfoor, G. H. Abdullah, F. Dworkowski, M. Wilson, D. Heyes, S. Hardman, L.
Murphy, R. W. Strange, N. S. Scrutton, C. R. Andrew and M. Hough , Chem. Sci., 2016, DOI:
Proximalvsdistalheme-NOcoordinationisanovelstrategyforselectivegasresponseinheme-basedNO-sensors.InthecaseofAlcaligenesxylosoxidanscytochromec'(AXCP),formationofatransientdistal6cNOcomplexisfollowedbyscissionofthetransFe-Hisbondandconversiontoaproximal5cNOproductviaaputativedinitrosylspecies.HereweshowthatreplacementoftheAXCPdistalLeu16residuewithsmallerorsimilarsizedresidues(Ala,ValorIle)trapsthedistal6cNOcomplex,whereasLeuorPhe residues lead toaproximal5cNOproductwitha transientornon-detectabledistal6cNOprecursor.Crystallographic,spectroscopic,andkineticmeasurementsof6cNOAXCPcomplexesshowthatincreaseddistalsterichindranceleadstodistortionoftheFe-N-Oangleandflippingoftheheme7-propionate.However,itisthekineticparametersof thedistalNO ligandthatdeterminewhether6cNOorproximal5cNOendproductsare formed.Ourdatasupporta‘balanceofaffinities’mechanisminwhichproximal5cNOcoordinationdependsonrelativelyrapidreleaseofthedistal NO from the dinitrosyl precursor. This mechanism, which is applicable to other proteins that form transientdinitrosyls,representsanovelstrategyfor5cNOformationthatdoesnotrelyonaninherentlyweakFe–Hisbond.Ourdatasuggestageneralmeansofengineeringselectivegasresponseintobiologically-derivedgassensorsinsyntheticbiology.
The molecular mechanisms that underpin heme proteindiscrimination between diatomic gas ligands (NO, CO andO2) are of fundamental importance in cellular respiration,signalling and toxicity defence and are thus of widerelevance. Understanding the means by which hemeproteins areable toproduce selectivity anddiscriminationin theirbindingof gases is vital for theeffectivedesignoradaptationofheme-basedsensorsforbiotechnology.Manypenta-coordinate heme proteins, for example thearchetypal myoglobin, simply bind gases to their vacantsixth coordination position at the distal heme face.However, in an increasing number of proteins it has beenshown that the key signalling molecule nitric oxide (NO)
formsafive-coordinatecomplexwithNO(5cNO)boundtothe proximal face of the heme. First identified incytochrome c’ from Alcaligenes xylosoxidans (AXCP)1,proximal NO binding has recently been confirmed also tooccurintheNO-activationmechanismoftheeukaryoticNOsensor soluble guanylate cyclase (sGC)2,3, bacterial hemenitricoxide/oxygenbinding(H-NOX)gassensors4-6,andthepro-apoptotic cytochrome c/cardiolipin complex7. Thefactors that control distal vs proximal heme-NOcoordination in these proteins are therefore of particularinterest.
Cytochromes c’ occur in methanotrophic, denitrifying andphotosynthetic bacteria and have proposed roles inprotection against nitrosative stress, NO trafficking duringdenitrification or pathogen defence8. All cytochromes c’havea4a-helixbundlestructurecontainingahemecentrewith a solvent exposed proximal His ligand and a buriedhydrophobic distal pocketwith a non-coordinated residue(LeuorPhe, rarelyMetorTyr) inapositiontoexertstericinfluence on the binding of diatomic gases. Unusually,cytochromes c’ are able to utilize both heme faces (distalandproximal)asameansofdiscriminatingNOfromotherdiatomicgases.Studiesofheme-NO-bindinginAXCPreveal
amulti-stepdissociativemechanisminwhichformationofadistal six-coordinate heme-nitrosyl (6cNO) complex isfollowed by scission of the trans Fe-His bond andconversion to a proximal 5cNO product via a putativedinitrosyl species (Scheme 1)9. While such dinitrosylcomplexes are transient in proteins, they have also beenexperimentally characterised in small molecule porphyrincomplexes10. In heme proteins, 5cNO formation has been traditionallylinked to an inherently weak Fe–His bond, which upondistal NO binding, facilitates His ligand release via anegativetranseffect.However,cytochromesc¢areunusualbecause they form 5cNO complexes despite relativelystrongFe–Hisbonds,withn(Fe–His) frequencies (~230cm-
1) significantly higher than the cut off limit of ~216 cm-1beyondwhichhemeproteinsarepredictedtoremaininthe6cNO state11.Moreover, the fact that the L16A variant ofAXCP (which forms only a distal 6cNO state) has a similarn(Fe–His) frequency to that of wt AXCP (which forms aproximal5cNOproduct) stronglysuggests thatdistal6cNO® proximal 5cNOconversion is governedby factorsotherthantheFe–Hisbondstrength12.
Scheme 1: AXCP Heme-NO Binding Mechanism and the Effect of Distal Leu16MutationsonObservedIntermediatesandProducts.
12-13witha6cNO distal complex being trapped and no proximal NOformation. In order to understand the factors controllingdistalversusproximalNOcoordination,wehaveexaminedNO binding to AXCP variants inwhich the occluding distalresidue Leu16 is replaced with residues that are smaller(Ala,Val),ofcomparablesize(Ile)or larger(Phe).Thedataare consistent with a ‘balance of affinities’ mechanismwherethedistalpocketoccludingresidueaffectsthekinetic
parametersk6onandk6offforNObindingandreleaseatthedistal heme face. The ratio of distal vs proximal faceaffinities determines which NO dissociates from thetransient dinitrosyl intermediate, leaving either a distal6cNO complex (L16A, L16V, L16I) or a proximal 5cNOcomplex(wtAXCP,L16F).Ourmechanismprovidesanovelroute for 6cNO® 5cNO conversion that does not requirean inherently weak Fe–His bond. It is the stericenvironment that underpins the reactivity differencesbetween distal and proximal sites. The same balance ofaffinities mechanism (perhaps involving other structuralproperties) could operate in any naturally occurring or
engineered heme-based NO sensor that generates atransientdinitrosylspecies.
All AXCP variants studied (L16A, V, I, and F) exhibit Fe(II)hemeabsorptionspectrasimilartothatofwtprotein14(Fig.S1a†). Then(Fe–His) frequenciesof Fe (II) L16V (234 cm-1)and Fe (II) L16I (235 cm-1) from room-temperatureresonance Raman (RR) spectra (Fig. S1†, Table S1†)resemblethoseofL16A(230cm-1)andwtAXCP(231cm-1)andsignifyarelativelystrongproximalbond.Onadditionofexcess NO, the L16F variant forms a 5cNO product (lmax
~395nm) similar to that of wt AXCPwhile the L16A, L16Vand L16I variants instead form only a 6cNO product (lmax~415nm)(Fig.S2†).Crystalstructuresweredeterminedforthe ferrousand ferrousnitrosyl complexesof thevariants,withredoxandligandstatesvalidatedusing insitu,onaxissinglecrystalresonanceRaman(SCRR)spectroscopy(Tables1 & S1-S3†, Figs. 1, S3&S4†) using methods previouslydescribed14-15.AllofthetertiarystructuresaresimilartowtAXCP and only salient points concerning the heme regionare discussed here. Ferrous structures show the differentresidues at position 16 to lie over the heme in a similarmanner to that of Leu16 in wt AXCP (Fig. S4†) with theexception of L16A where the side chain is essentiallytruncated.While the structures of ferrous L16V, L16I, andwt AXCP have empty distal sites, in L16A a well-definedwater ligand (not observed in solution RR spectra) ispresentatadistanceof2.17Å.(Fig.S4†). Insharpcontrasttotheproximal5cNOproductformedbywtAXCP(Fig.1a), thestructureof ferrousnitrosylL16A(Fig.1b, Fig. S5, Table1) confirms thepresenceof adistal6cNO complex as suggested by spectroscopic data12. Thedistal NO ligand at full occupancy does not form anyhydrogenbondstoproteinresiduesandtheFe-N-Oangleis135°withnoindicationofstericconflictbetweentheAla16residueandNO.SubstitutionoftheoccludingLeu16residuein the distal pocket by the smaller Ala thus results in NObinding essentially without steric effects from the proteinenvironment andwith Fe-N-O geometry similar to that of{FeNO}7model complexes. Consistentwith the removal ofdistal steric hindrance, the Leu16®Ala mutation isassociated with a ~100-fold increase in k6on (vide infraand12-13).
Compared to L16A, the L16I AXCP variant experiencesgreatersterichindrancetowardsdistalNObinding(Fig.1c,Fig. S5). The crystal structure of the 6cNO L16I complexshowstwoorientationsofNO,orientedtowardsMet19andPro55 respectively. The Fe-N-O angles for these are verysimilar (136°and139°)andonlyoneconformerof Ile16 ispresent. Ile16undergoesamodestshift inposition(~0.9Å)uponbindingofNOwhilemaintainingasimilarrotamer.
Fig.1.2Fo-Fcelectrondensitymapsoftheferrous-nitrosylcomplexesinwtAXCPanddistalvariants(contouredat1.0σ).(A)Proximal5cNOcomplexinwtAXCP.(B) Distal 6cNO complex of L16A (C). Distal 6cNO complex of L16I with thepresenceoftwoNOconformersandoneIle16conformationbuttheabsenceofthe7-propionateflip.(D)Distal6cNOcomplexofL16VwiththepresenceoftwoNO and Val16 conformers and a partial 7-propionate flip. (E) Proximal 5cNOcomplexinL16F.(F)Superpositionofthedistal6cNOcomplexintheL16Vvariant(purple)and thedistal6cCOwtAXCPstructure (blue).Fo-Fcomitmaps forNOcomplexesareshowninFig.S5.
In this structure, theheme7-propionate is not flipped to theproximal pocket (as observed in the 6c-CO complex of wtAXCP)butdoeshaveanalteredconformation.RelativetoL16I,theL16Vvariantexhibitsmoreextensiverearrangementsupon6cNO formation.Althoughsmaller than Ile, theorientationofthe Val side chain in the L16V variant brings it closer to thehemepropionates (Fig. 1d, Fig. S5) and there is a smallmainchainshiftoftheValawayfromMet19.
TwoNOorientationswithpartialoccupanciesarealsopresentin L16V. In one of these, NO is positioned towards the sidechain ofMet19 and the Fe-N-O angle is 127°. In the secondconformation, NO is oriented towards Trp56 and the Fe-N-Oangle is 132°. In the 132° conformer (but not the 127°conformer), the Val16 conformation has undergone a ~180°rotation, causing it to occupy a position where it wouldprovokeastericclashwith theorientationof thedistalheme7-propionate (3.37 Å compared to 4.20 Å) in the 5c ferrousstructure,causingthepropionatetofliptowardstheproximalpocket (Fig. 1d, Fig. S5). These structural rearrangements areanalogoustothoserecentlyobservedforthe6cCOcomplexofwtAXCP,inwhichanear-linearFe-C-Ogeometryforcesa120°rotation of the Leu16 Ca–Cb bond together with a distal toproximal flipof the7-propionate.A secondconformerwithabent Fe-C-O (158°) is associated with smaller movements ofLeu16andnopropionateflip13.ThereasonwhyaFe-C-Oangleof 158° does not lead to a propionate flip, whereas a morecompressedFe-N-Oangleof132° doesmay indicate that theorientation of the Fe-X-O unit (not just the bond angle) isimportant.
A superposition of the wt AXCP 6cCO structure with theL16V6cNOstructureisgivenin(Fig.1f).Whilethestructureofthe transient 6cNO complex of wt AXCP has yet to bedetermined, the structural rearrangements observed uponL16V 6cNO and wt 6cCO formation suggest that distal NObindingtowtAXCPisalso likelyto involvesignificantrotationof the Leu16 residueandaproximal flipof the7-propionate.Further insights into the 6cNO structure are provided by RRspectroscopy (vide infra). Finally, the crystal structure of theL16F variant shows two equally occupied proximal 5cNObinding modes (Fig. 1e, Table 1), similar to the proximalcomplex previously observed for the wt AXCP end product,and isnotdescribed indetailhere. Kineticdataconfirm thatthis variant has a high degree of distal steric constraint thatdestabilizesdistalheme-NObinding(videinfra).
RRspectraof6cNOAXCPcomplexesyieldcomplementarystructural information on the heme-NO environment insolution(Figs2,S6-S8†).Porphyrinmarkervibrationsoffrozensolutions (100K)have frequencies typicalof6cNOheme(Fig.S6,Table2),andaresimilar (±5cm-1) tothoseobtainedfromsinglecrystals(Fig.S3†,Table2).
aFrequencies are from RR spectra of protein solutions at pH 7.0, or from single crystals at pH 7.5 (data in italics) except for bStopped-flow FTIR data at pD 9.4.Abbreviations:rt;roomtemperature,sw;spermwhale,cthiswork.
The n(N–O) stretching frequencies of L16A (1630 cm-1),L16V,(1626cm-1),andL16I(1621cm-1)are identifiedfromtheir~25-cm-1downshiftswith15NO(Fig.S5†)ora~70-cm-1downshiftwith15N18O(Fig.S7†).TwobandswithmixedFe-NO stretching/bending character are also evident in the~450–460 cm-1 and ~560–580 cm-1 regions, denotedn(FeNO)I and n(FeNO)II respectively (Fig. 2). Althoughvibrational assignments of 6cNO complexes have beencontroversial20-22, recent nuclear resonance vibrationalspectroscopy (NRVS) studies point to n(FeNO)II as thepredominant Fe-N-O bend and ν(FeNO)I (not alwaysobserved in RR spectra) as the predominant Fe-NOstretch23-25. Then(FeNO)IImodes of L16A (568 cm-1), L16V(578 cm-1), and L16I (578 cm-1) are readily identified fromtheir ~16-cm-1 downshifts with 15NO (Fig. 2) or ~20-cm-1downshiftwith 15N18O (Fig. S7†),while the relativelyweakn(FeNO)I bands of L16A (454 cm
-1) , L16V (454 cm-1), andL16I (459 cm-1) – obscured by an overlapping porphyrinmode – are identified from ~6-cm-1 downshifts with 15NO(Fig. 2) or a ~10-cm-1 downshift with 15N18O (Fig. S8†). Aspreviously observed for Rhodobacter capsulatuscytochromec' (RCCP)18,6cNORR frequenciesare sensitivetosampletemperature(Table2). Since all of the 6cNO AXCP variants have hydrophobicdistal pockets (Fig. 1), it appears that the variations in RRfrequencies arise from steric rather than electrostaticeffects. DFT calculations by Spiro and co-workers predictthat compressionof theFe-N-Oanglebelow~140° shouldlower the n(N–O) frequency23, in agreement with ourcrystallographicandRRdata.Ontheotherhand,decreasingthe Fe-N-O angle is also predicted to weaken the Fe–NObond, whereas we observe that both n(FeNO)I andn(FeNO)IIareatsimilarorhigherfrequenciesinsterically
Fig. 2. Low-frequency RR spectra of 6cNO AXCP solutions (100 K) obtainedwith406.7nmexcitation:(A)L16A,(B)L16V,and(C)L16Iproteinspreparedwith14NO(black)and15NO(red).Isotopedifferencespectra(blue)identifythen(FeNO)Iandn(FeNO)II vibrations. The L16A n(FeNO)I frequency is identified from a largerisotopeshiftwith15N18O(FigureS8†).
6cNOprotein
Temp n4 n3 n2 n10 n(FeNO)I n(FeNO)II n(NO) ref
AXCP (L16A) rt 1373 1500 1593 1631 454 563 1630 c 100K 1373 1501 1595 1635 454 568 1631 c 100K 1372 1501 1592 1632 c (L16V) 100K 1374 1501 1595 1634 454 578 1626 c 100K 1377 1597 1632 c (L16I) 100K 1374 1501 1595 1634 459 578 1621 c 100K 1372 1592 c (wt) rt 1625b 16 90K 1375 1504 1596 1638 579 1624 17
constrained sites (Table 2). This discrepancy may reflect thedifficultyofmodellingtheangulardependenceofthesemixedvibrationalmodes26.
Wealsonotethatcorrelationsbetweenspectroscopicandstructural data could be affected by conformationaldifferences between the crystalline and solution state. Forexample,theL16VandL16IstructuresexhibitmultipleFe-N-Oconformers,whereasthere isnoevidenceformultiplesetsofheme-NO RR bands in solution. Nevertheless, comparison ofthe present (100 K) RR datawith previousmeasurements onthefrozenwt6cNOAXCPintermediaterevealstheinfluenceofdistal steric constraints on heme-NO vibrational frequencies.Most notably, the sterically constrained L16V and L16I 6cNOcomplexesexhibita~10cm-1upshiftinn(FeNO)IIanda~5–10cm-1 downshift in n(NO) frequencies relative to L16A.Importantly, the RR frequencies of the L16V and –I variantsresemble those of the transient wt 6cNO complex, implyingthatthestructuresoftheheme-NOchromophoresaresimilar(despite differences in 6cNO stability). This suggests thatdistortionoftheheme-NOunitisnottheultimatedeterminantof distal 6cNO® proximal 5cNO conversion. Instead, ourstudiessuggestthatdistal6cNOvsproximal5cNOformationisdeterminedbythekineticpropertiesofthedistalandproximalhemefaces(videinfra).
We used stopped-flow optical spectroscopy to understandhow the distal L16 mutations and the consequent structuralchanges in the distal pocket perturbed reactivity. Kineticconstants obtained are summarized in Table 3 and Fig. 3. Aspreviously observed9,17, wt AXCP undergoes an initial distalNO-binding event to form an observable 6cNO intermediate(k6on)whichsubsequentlyconvertstoaproximal5cNOproduct(k6-5) (Fig. S9†). The L16F variant forms a proximal 5cNOcomplex inasimilarmannertowt,albeitwithchangestothekineticparameters(Fig.S9†,Table3).Bycontrast,theL16V,-A,and–Ivariantsformstabledistal6cNOproducts(Fig.S10†)
without the ‘distal-to-proximal’ conversion exhibited by wtandL16FAXCP(Fig.S9†).Previouskineticdatarevealedthatk6onforL16A(2.9´10
6M-
1s-1)increasedbytwoordersofmagnituderelativetowtAXCP(4.33´ 104M-1s-1) (Table 3)12-13. Herewe show that the k6onvaluesforL16V(1.52±0.03´106M-1s-1)andL16I(1.79±0.15´106M-1s-1)aremidwaybetweenthoseofL16AandwtAXCP,
hindrance, thevaluesofk6offprogressively increase.Thus, theoverall effect of distal steric constraints on KD values(calculated from the k6off/k6on ratio) is to lower the distalheme-NOaffinity.
In order to characterize the influence of the AXCP distalpocket structure on geminate recombination we carried outtime-resolved infra-red (TRIR) experiments for NO rebindingfollowing laser flash photolysis, (Figs. 4, S13†). In previousstudies on wt AXCP, geminate rebinding of a population ofdistal 6cNO (generated by addition of sub-stoichiometricamountsofNO)wasdeterminedtooccurwithatimeconstantof 52 ps27.Our TRIRmeasurements for the 6cNO complex ofL16I and L16V indicate the rebinding of NO at the distal sitewith time constants of 6.8±0.95 ps (L16I) and 7.4±1.53 ps(L16V)withanadditionalslowerphasewithtimeconstantsof52 ps (L16I) and 364 ps (L16V) (Fig. S13†). TRIR data for theL16A variant have been described previously28. Faster and/ormore complete geminate recombination in variants withsmallerdistalpocketresiduescouldcontributetotheobservedchangesink6off(NO).Futurestudiesoverextendedtimeframeswill probe geminate NO rebinding to AXCP variants in moredetail, including the influence of distal residue and heme 7-propionate rearrangements. Indeed, recent moleculardynamics simulations of geminate CO-rebinding in wt andL16A AXCP suggest that propionate conformation is a keydeterminantofdistalAXCP-ligandaffinity29.
Ourdataprovide insights into thekeyquestion:whydoeswtAXCP undergo a distal 6cNO® proximal 5cNO conversion(involving Fe-His scission) whereas L16A, L16V and L16Ivariantsremain6cNO?NotonlydoesAXCPnotpossessaweakFe–Hisbond,thepresentstudysuggeststhatadistorteddistalFe-N-Ogeometryand/orflippingoftheheme7-propionatearenot sufficient in themselves to drive proximal 5cNOconversion. Instead, kinetic trends show that proximal 5cNOformation occurs when the distal NO ligand of the 6cNOcomplexhasahigherk6offandalowerk6onvalue(Fig.3,Table3).
Distal6cNO®proximal5cNOconversioninvolvesbreakingthe Fe–His bond to form a putative distal 5cNO, followed byattack of a proximal NO to form a transient dinitrosyl andfinally the release of the distal NO to generate a proximal5cNOproduct (Scheme1).Wepropose that the initial Fe–Hisbond scissionby the trans effect is similar forwt and variantproteins, irrespective of the distal pocket occluding residue.Because of their relatively strong Fe-His bonds, we proposethat the 6cNO species is in equilibrium with only a traceamount of distal 5cNO (below detection limits). Subsequentreaction of the distal 5cNO population with a second(proximal)NOgenerates a transientdinitrosyl.Assuming thatthedinitrosylspeciesexhibittrendsindistaloffratessimilartothoseofthe6cNOspecies(Fig.3),ourdatastronglysupportabalance of affinities mechanism where a high distal k6offrelative to proximal koff in the dinitrosyl precursor traps the
proximal 5cNO product in wt AXCP (and also in L16F). Invariantswheredistal6cNOistrapped(L16A,-V,-I),thevaluesof k6off are much smaller than that of wt AXCP while theproximalvaluemayreasonablybepresumedtobeunchanged.In these variants, proximalNOpreferentially dissociates fromthedinitrosyl complex and theproximalHis ligand rebinds toformtheexperimentallyobserved6cNOcomplex.
Several heme proteins, including cytochromes c’, exhibitkineticbehaviourconsistentwithproximal5cNOformationvia6cNO and putative dinitrosyl precursors. We propose thatthese proteins may share a common kinetic ‘balance ofaffinities’mechanism. Ineachprotein, initialbindingofNOtothedistalhemefacecausesHisliganddissociationviathetranseffect, followed by the binding of a second NO to form thedinitrosyl intermediate. The balance of affinities determineswhichofthetwoNOligands inthedinitrosyldissociatesfromthe Fe and hencewhich adduct is eventually formed.Wheretheaffinityon theproximal side ishigher, theproximal5cNOformpredominates,whereaswhenthedistalaffinityishigher,rapidHisreattachmentleadstoadistal6cNOproduct.Kineticdata have revealed NO-dependent 6cNO®5cNO conversion(consistent with proximal 5cNO formation via a transientdinitrosyl) in sGC,H-NOX fromVibrio cholera (VcH-NOX)4,H-NOX from Clostridium botulinum (Cb H-NOX)6, and in thecardiolipin/cytochrome c complex7, although we note thatstructural data are not yet available for these complexes.Althoughk6onvalues for the6cNO intermediatesofVcH-NOX,CbH-NOXand sGCproteins are relatively high (³ 108M-1 s-1)andconsistentwithalowdegreeofdistalsterichindrance,thecorresponding distal 6cNOk6off values are also relatively high(0.3, 0.012, and 27 s-1, respectively), such that the putativedinitrosyl species might foreseeably decompose bypreferentialreleaseofdistalNOinamanneranalogoustothatproposedforAXCP.
ConclusionsOur data show that 6cNO binding to the distal heme face ofAXCP predominates when residue 16 is small (Ala, Val, Ile),evenwhensignificantdistortionoftheFe-N-Oangleispresentand where a flip of the heme 7-propionate has occurred. Incontrast, when Leu or Phe are present in the occludingposition, the 6cNOdistal complex is an intermediate prior toformation of a proximal 5cNO species. Taken together, ourdata are consistent with a ‘balance of affinities’ mechanismwhere the distal pocket occluding residue affects the kineticparameters k6on and k6off for NO binding to the distal hemeface.TheratioofdistalandproximalfaceaffinitiesdetermineswhichNOfromthetransientdinitrosyl[6c-(NO)2]intermediatedissociates, leavingeitheradistal6cNOcomplex (L16A,L16V,L16I) or a proximal 5cNO complex (wt AXCP, L16F).Modificationofdinitrosyldynamicsrepresentsanovelstrategyfor modulating heme-NO response by controlling distal vsproximal heme-NO coordination as well as 6cNO vs 5cNOcoordinationgeometry.
The preparation of recombinant wt and L16A AXCP weredescribed previously13,15. To generate the L16V, L16I & L16Fvariants, a modified Quikchange site directed mutagenesismethod was applied to the AXCP gene in plasmid pet26b(+)using primers shown in ESI†. Protein expression, purificationand crystallisation were as described previously15 as wereprocedurestoremoveanyendogenouslyboundgasligands.Toreducecrystalstotheferrousstatetheyweretransferredinto~2mlofdeoxygenatedbuffercontaining100mMascorbateina supasealed glass vial for ~3h. To generate the NO-boundstate, 10 µl of 80 mM stock of the NO donor compoundproliNONOate was injected through the supaseal. Crystalswere transferred using a cryoloop into cryoprotectantcomprising 40% sucrose, 2.4 M ammonium sulfate, 100 mMHEPESpH7.5for~10sbeforetransferintoliquidnitrogen.
X-raydatacollectionandprocessing
All crystallographicandsinglecrystal spectroscopicdataweremeasured at the Swiss Light Source, beamline X10SA. X-raydiffractiondataweremeasuredusingaPilatus6M-Fdetectorand processed using XDS30. Data reduction and refinementwere carried out in the CCP4i suite using AIMLESS31 andREFMAC532 with the most appropriate AXCP structure fromthe PDB chosen as the starting model. Between cycles ofrefinement the structures were rebuilt in Coot33. Thestructures were validated using the JCSG QC server andMolprobity34. On convergence of Refmac5 refinement,structures with resolution 1.25 Åor higher were furtherrefined in Shelxl35 to obtain estimated standard uncertaintiesforbondlengthsandangles.Fo-FcomitmapsweregeneratedtoguideandvalidateligandmodellinginstructureswhereNOwasobservedintwoalternatepositionswithpartialoccupancy(Fig. S5†). Coordinates and structure factors have beendeposited in the RCSB Protein Data Bank. Data collection,processingandrefinementstatisticsareshownin(TableS2†).
SCRR spectra were measured from the crystals used forstructure determination with the MS3 on-axismicrospectrophotometeratbeamlineX10SA36witha405.4nmexcitationlaser.SpectraweremeasuredpriortoandfollowingX-ray data collection to check for changes to the samplecausedbytheexcitationlaserorX-rayradiolysis.Ramanshiftswere calibrated using cyclohexane or paracetamol as areference. The laser powers at the sample position wereselected to be below the threshold of laser-induced photo-reductionandwereintherangeof0.88to4.30mW.TheSLS-APEtoolbox37wasusedtoanalyseallSCRRdata.
ResonanceRamanspectroscopy
Ferrous L16 variants and their complexes with NO wereprepared in an anaerobic glove box. Protein was reduced totheferrousstatewithexcesssodiumdithionite.ToprepareRRsamples of gaseous heme complexes, excess dithionite was
removed using a minispin desalting column (Zeba filter,Pierce),followedbyintroductionofgas intotheheadspaceofseptum-sealed capillaries using a gas-tight Hamilton syringe.TheidentityofRRsampleswasverifiedbyUV-visspectroscopybeforeandafterexposuretothe laserbeamusingamodifiedCary 50 spectrophotometer. RR spectra were recorded on acustom McPherson 2061/207 spectrograph (set to 0.67 m)equipped with a Princeton Instruments liquid N2-cooled (LN-1100PB)CCDdetector. Excitationwavelengthswereprovidedby the406.7nmand413.1nm linesofaKr ion laserandthe441.6 nm line of a He-Cd laser. Rayleigh scattering wasattenuatedusingsupernotchfilters(Kaiser)orlong-passfilters(RazorEdge, Semrock). RR spectra of frozen samples,maintained at 100 K with a liquid nitrogen cold finger, wereobtained using a ~150° backscattering geometry and laserpowers of 5 – 25 mW (at the sample). A 90° scatteringgeometry was used for RR spectra of room temperaturesamples.RRspectraweretypicallymeasuredforperiodsof2–5minwithindeneandaspirinusedtocalibrateRamanshiftstoanaccuracyof±1cm-1.Stopped-flowkineticsofNObindingandrelease
The SX-20 UV-visible stopped-flow spectrophotometer(Applied Photophysics) with a diode-array detector was usedto record the reaction of ferrous protein with NO at 25 °C.Spectrawereobtained from360 to700nmwithadead-timeof ~1.3 ms. A 40-fold excess of sodium dithionite solution(~200 μM) was used to prepare the ferrous proteins indegassed buffer (50mMCHES and 100mMNaCl, pH 8.9) tomatch previous experiments. The NO donor proliNONOate(dissolvedin1mLof25mMNaOH)withastockconcentrationof ~80 mM was diluted into the degassed buffer (pH 8.9)yieldingthedesiredconcentrationsoffreeNO. The concentrations of dissolved NOweremaintained at a~10-fold excess over the heme binding sites (~5 μM aftermixing) to ensure pseudo-first order conditions. FerrousproteinsolutionsweremixedwithNO-containingbufferintherange of 50 – 1850 μM (after mixing). Reactions weremonitoredusingmonochromaticlightat393,416and436nmusingaphotomultiplierdetectorand from360–700nm(0–500 s) using a photodiode array detector. Pseudo-first-orderrate constant at each [NO] was determined by fittingexponential timecoursesusinga least-squares fittingmethodand plotted against the [NO] to yield the second-order-rateconstant.TheGlobalAnalysisofmulti-wavelengthkineticdatawas carried out using the Pro-Kineticist software package(AppliedPhotophysics).
The release ofNO from L16V and L16I AXCPwas initiated byreacting the nitrosyl complexwith a solution of 14 – 59mMsodium dithionite in the presence of ~0.5 mM CO in ananaerobic cuvette. Time-resolved UV-vis absorption spectra,recorded at 25 °C using a Cary 60 spectrophotometer, wereused to monitor the rate of disappearance of the 6cNOcomplexviatheappearanceof6cCOabsorbancefeatures(lmax
418nm).Rate constants forheme-NO release (k6off)obtainedfrom exponential fits of the 418 nm absorbance time coursewereinsensitivetovariationsindithioniteconcentration.Time-resolvedinfra-redspectroscopy
Time-resolved infra-red (TRIR) experiments were carriedout as described previously28 using the ULTRA instrument,Central Laser Facility, Rutherford Appleton Laboratory. WTAXCPandthedistalL16Iand–Vvariants(5μM)werepreparedanaerobicallyinaglovebox.Theferrousstateswereachievedbytheadditionofsodiumdithionite (fewgrains)andtheNO-bound states were achieved by the addition of 1mgproliNONOatepowderfollowedbyanincubationof~1hourat20°C.SampleswerecontainedinacellwithCaF2windowsanda 75 µm pathlength. The cell was rastered to avoid photo-bleaching. The laser excitation wavelength was 532 nm anddata were collected between time delays of 0.0005 - 1 nswithin a spectral window of 1300 - 1800 cm-1 using twooverlapping 128 pixel detectors. The resolution of theinstrument is ~3 cm-1 pixel-1 and pixel to wavenumber wascalibrated using polystyrene. In-house Ultraview (version 3)software was used to process the data and the kineticparameterswerefittedusingOriginPro.
AcknowledgementsWegratefullyacknowledgetheroleofDr.SvetlanaAntonyuk,Professor Robert R. Eady and Professor S. Samar Hasnain oftheMolecularBiophysicsGroup,UniversityofLiverpoolwheretheearlypartofthisworkbeganandforongoinginsightsanddiscussions. We also acknowledge the contributions of NeilRustage andDr.Mark Ellis in the early stages of this project.C.R.A. acknowledges support from the National ScienceFoundation (grantsMCB-0745035andMCB-1411963) andDr.PierreMoënne-Loccoz for assistance with RRmeasurements.D.G. & G.A. were hosted for this work by the MolecularBiophysicsGroup,UniversityofLiverpool.Thisworkwaspartlysupported by EPSRC grant EP/J020192/1 to N.S.S. D.K. wassupportedbyaSchoolStudentshipat theUniversityofEssex.G.A.’sPhDStudentshipwassupportedbyBangorUniversity.X-ray diffraction dataweremeasured at the Swiss Light Sourceunder long-termaward20111166 toM.H.and funded inpartby the EU FP7 programme via BioStructX awards 2370 and6714 toM.H& R.W.S.We acknowledge the assistance of DrAndreyLebedevwithrestraintlibrariesinRefmac5refinement.
References1 D.M Lawson, C. E. M. Stevenson, C. R. Andrew, and R. R.
Eady,EMBOJ.2000,19,5661-5671.2 A-L Tsai, V. Berka, I. Sharina and E. Martin. J. Biol. Chem.
2011,286,43182-43192.3 A.P Hunt and N. Lehnert,Acc. Chem. Res., 2015, 48, 2117-
8 M. A. Hough and C. R. Andrew, Advances in MicrobialPhysiology. Recent advances in microbial oxygen-bindingproteins;PooleR.K.,Ed.;Elsevier:UK,2015,67,1-84.
9 D. A. Pixton, C. A. Peterson, A. Franke, R. van Eldik, E. M.GartonandC.R.Andrew,J.Am.Chem.Soc.,2009,131,4846-4853.
12 E.M. Garton, D. A. Pixton, C. A. Peterson, R. R. Eady, S. S.Hasnain and C. R. Andrew, J. Am. Chem. Soc., 2012, 134,1461-1463.
13 S. Antonyuk, N. Rustage, C. A. Peterson, J. L. Arnst, D. J.Heyes, R. Sharma,N. Berry,N. S. Scrutton, R. R. Eady, C. R.AndrewandS. S.Hasnain,Proc.Natl.Acad.Sci.USA., 2011,108,15780-15785.
14 C. R. Andrew, E. L. Green, D. M. Lawson and R. R. Eady,Biochemistry,2001,40,4115-4122.
15 D.Kekilli,F.S.N.Dworkowski,G.Pompidor,M.R.Fuchs,C.R. Andrew, S. Antonyuk, R. W. Strange, R. R. Eady, S. S.HasnainandM.A.Hough,ActaCryst.,2014,D70,1289-1296.
16 S. J.George,C.R.Andrew,D.M.Lawson,R.N.F.ThorneleyandR.R.Eady,J.Am.Chem.Soc.,2001,123,9683-9684.
17 C. R. Andrew, S. J. George, D. M. Lawson and R. R. Eady,Biochemistry,2002,41,2353-2360.
18 C.R.Andrew, L. J.Kemper,T. L.Busche,A.M.Tiwari,M.C.Kecskes, J. M. Stafford, L. C. Croft, S. Lu, Pierre Moënne-Loccoz, W. Huston,‖ J. W. B. Moir‖and R. R. Eady,Biochemistry,2005,44,8664-8672.
25 N. Lehnert, J. T. Sage,N. Silvernail,W. R. Scheidt, E. E. Alp,W.SturhahnandJ.Zhao,Inorg.Chem.,2010,49,197-215.
26 N. Lehnert, W. R. Scheidt and M. W. Wolf, Struct. Bond.2014,154,155-224.
27 B. K. Yoo, I. Lamarre, J. L. Martin, C. R. Andrew and M.Negrerie,J.Am.Chem.Soc.2013,135,3248-3254.
28 H.J.Russell,S.J.O.Hardman,D.J.Heyes,M.A.Hough,G.M.Greetham, M. Towrie, S. Hay and N. S. Scrutton. FEBS J.2013,280,6070-6082.
29 C. R. Andrew, O. N. Petrova, I. Lamarre, J.-C. Lambry, F.Rappaport and M. Negrerie, ACS Chem. Biol. 2016, justaccepted(DOI:10.1021/acschembio.6b00599)
30 W.Kabsch,ActaCryst.,2010,D66,125-132.31 P. R. Evans and G. N. Murshudov, Acta Cryst., 2013, D69,
1204-1214.32 G.N.Murshudov,A.A. Vagin and E. J.Dodson,ActaCryst.,
1997,D53,240-255.33 P. Emsley, B. Lohkamp, W. G. Scott and K. Cowtan, Acta
