Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping

Article abstract of DOI:10.1021/jacs.9b05715

Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and t

Full text of DOI:10.1021/jacs.9b05715

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Article
Tuning radical relay residues by proton
management rescues protein electron hopping
Estella F Yee, Boris Dzikovski, and Brian R Crane
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b05715 • Publication Date (Web): 11 Oct 2019
Downloaded from pubs.acs.org on October 13, 2019
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Scheme 1 – Reactions of WT CcP:Cc. Protonation of the ferric hydroxide in step III may lag or precede
reduction of W191•+ in step IV depending on conditions.
1
53x22mm (300 x 300 DPI)
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Figure 1 — Cc oxidation reactions under multiple turnover conditions. (A) Oxidation of Cc(Fe2+) by CcP
variants was measured at Abs550 nm−Abs540 nm, with values normalized from 0 to 1 (100 mM potassium
phosphate (KPi), pH 6 for all variants except for W191Y:L232H, which was at pH 7). (B) Progress curves at
Abs434 nm, reflect formation and decay of the ferryl (Fe4+=O) species. (Curves were normalized and
shifted vertically).
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Figure 2 — Correlation between single and multiple turnover rate constants for Cc oxidation. (A) Single
turnover rate constants obtained in 100 mM KPi buffer, pH 6 (Supplemental Table 1). (B) Multiple turnover
rate constants in 100 mM KPi buffer, pH 6 (Supplemental Table 1). (C) Correlation between single and
multiple turnover rate constants indicates that Cc oxidation is not dominated by protein exchange kinetics in
the multiple turnover format.
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Figure 3 – Conjugate base next to Tyr191 in CcP alters the pH dependence of Cc oxidation. Kinetic traces of
Cc oxidation (lower curves) and CcP ferryl formation/decay (upper curves) were obtained with either (A)
W191Y CcP, (B) W191Y:L232E CcP, or (C) W191Y:L232H CcP. W191Y shows little variation with pH whereas
oxidation rates with either the Glu232 or His232 variant gives strong pH dependencies indicative of a
hydrogen bond to Tyr191. (D) Dependence of Cc(Fe2+) oxidation rates on pH for CcP W191Y:L232H in 100
mM KPi buffers. Fit to a two-proton ionization model (Equation 1) gives two pKa values of 6.46 and 7.24,
which likely coincide with the pKa of His and the hydrogen-bonded complex, respectively.
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Figure 4 – Hydrogen bond environments surrounding residue 191 in crystal structures of CcP:Cc complexes.
Fo – Fc difference maps of (A) W191Y CcP, (B) W191Y:L232E CcP, and (C) W191Y:L232H with the heme and
residues 191 and 232 omitted from the phase calculations. Maps are displayed at a contour level of 2 σ.
Distances are measured from the heme iron to the center of the phenol ring and between the Tyr191
hydroxyl and the residue 232 side chain atom of closest approach. Distances are average measurements
from the two complexes contained in the ASU. (D) Superposition of CcP Y191 variants: W191Y (red),
W191Y:L232E (purple), W191Y:L232H (green), all in complex with Cc. Substitution of Leu232 with Glu
results in a 1.04 Å displacement of Tyr191. Replacement with His232 does not significantly shift the position
of Tyr191; however, under these crystallization conditions, His232 is angled away from Tyr191, which is
likely a result of hydrogen bonding to Thr234 or an electrostatic interaction with Asp235.
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Figure 5 – cw-ESR of Cpd I provides evidence for hydrogen bond formation in L232E,H CcP. (A) X-band cw-
ESR spectra of Cpd I acquired at 12 K for CcP variants. Samples contained 100 mM KPi, pH 6 buffer, except
for where indicated. W191Y:L232H at pH 7.5 was acquired with 4 G modulation, and all others were
collected with 1.5 – 2 G modulation. Spectra were base-lined, normalized, and shifted vertically. (B) In the
X-band cw-ESR spectra of W191Y:L232E, high pH buffer results in narrowing and decrease of signal
intensity (inset), indicating loss of the phenolic oxygen hydrogen bond. (C,D) X-band cw-ESR of H2O and
D2O-exchanged Cpd I. W191Y Cpd I shows only modest changes after buffer-exchange, whereas
W191Y:L232E Cpd I exhibits a narrowed line shape in D2O that resembles that of W191Y Cpd I.
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Figure 6 – Three-pulse ESEEM spectra collected at 33.8 GHz on samples of W191Y Cpd I, W191Y:L232E Cpd
I, and photogenerated tyrosyl solution. (A,B) Normalized 1H-ESE-ENDOR spectra of W191Y and
W191Y:L232E Cpd I species and photogenerated tyrosyl in H2O and D2O 100 mM KPi buffer, pH 6; νLarmor
∼51 mHz. Subtracted spectra of Cpd I species (in dark and light grey) in protonated and deuterated
environments reveal H-bonding interactions. Spectral interference with other weakly bound protons,
however, makes quantification difficult. The asymmetric shoulder feature in the W191Y Cpd I difference
spectrum is likely not significant. (C) Time domain spectra (vertically stacked and amplitudes normalized
from 0 to 1 between 0 ns and 3000 ns) exhibit weaker stimulated-echo amplitude for W191Y Cpd I
compared to similarly prepared samples of W191Y:L232E Cpd I and photogenerated Tyr. (D) Fourier-
transformed signals show hyperfine couplings near that of 2H Zeeman frequency. Signals normalized to the
echo amplitudes at 0 ns demonstrate the lower levels of hydrogen bonding in W191Y Cpd I compared to
W191Y:L232E.
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Figure 7 – RFQ X-band ESR spectroscopy of Tyr• radicals in CcP. X-band ESR tyrosyl radical signals were
integrated and normalized to an Er3+ internal standard signal. Y191• was generated under single turnover
conditions by reacting 0.15 mM CcP with 0.3 mM H2O2 and quenched by 0.3 mM Cc(Fe2+). Integrated,
averaged, and normalized signals were fit by biexponential curves following a “competitive reaction” model
(
red and purple lines; Equation 6). Comparison between (A) W191Y and (B) W191Y:L232E demonstrate
differences in radical progression, which are reflected in the development of the ferryl species by UV-vis
kinetics.
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Figure 8 – Brønsted linear free energy plots to evaluate possible contributions of PCET, PTET, and ETPT on
Tyr191• reactivity; parameters are taken from Supplemental Table 3. Graph (A) shows poor correlation
between ΔΔG* derived from the rate constants and the ΔΔG°’ values calculated from Equation 3. In plot (B),
setting ΔE°’ = 0 produces a modest negative correlation. However, if ΔΔpKa = 0, there is good agreement
between the calculated changes, activation energy, and driving force (plot C), which indicates that the
system is primarily affected by changes in ΔE°’; proton affinity has little influence on the reactivity
differences of different species. The grey dashed curve illustrates a reasonable fit to the Marcus relationship
expressed by Equation 8 with �� ≣ 0.7 eV, E°’(W191Y CcP) ≅ 0.61 eV, and R2 = 0.7. (D) Plot of
reorganization energy isopleths (grey lines) as a function of driving force (between Cc(Fe2+) and Y191) and
driving force perturbation from the addition of E232 to Y191 CcP, as calculated from interprotein ET rates.
By Marcus theory, ΔΔG°^'=-ΔG°^'-λ+√(4λk_B Tln(k_1/k_2 )+(ΔG°^'+λ)^2 ), where the single turnover
reaction rate constants used are k1,obs = 0.05 s-1 and k2,obs = 0.19 s-1 for W191Y and W191Y:L232E
CcP, respectively; ΔG°’1 = E°’(Cc) – E°’(W191Y CcP); and –ΔΔG°’ is the change in formal potential of Y191•
CcP with E232. Reorganization energy λ is assumed to be similar between variants. The sign of the root was
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chosen for such that –ΔΔG°’ increases as –ΔG°’1 increases, i.e., the reaction is not in the Marcus inverted
region. The red square data points denote values of ΔΔG°’ for W191Y:L232E consistent with ΔG°’1
calculated from comparing single turnover rates between Y191 and CcP systems where –ΔΔG°’ is known.
Using the Marcus relation above, ΔG°’1 was calculated at different λ values and only plotted if there was
consensus with the calculated isopleths. Values are shown for rate data of F3Y191 CcP (kobs = 0.07 s-1).
This analysis provides a range of possible –ΔG°’1 and corresponding λ values for the Y191 CcP:Cc system,
giving an approximate –ΔΔG°’ between 0.18 and 0.21 eV.
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Figure 9 – (A) Schematic of stepwise ETPT (black) and PTET (grey) mechanisms for Tyr oxidation. Data
presented in Figure 8 support an ETPT pathway for Tyr• reduction by Cc(Fe2+) (grey; right to left). (B)
Changes in Tyr191• formal potential owing to substitution by non-natural FTyr or addition of a hydrogen
bond donor to the phenolic oxygen shown relative to the potentials of Cc(Fe2+)72 and
CcP(Fe4+)123,124,136.
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ꢀ
ꢀ
ꢀ
ꢀ
Tuningꢀradicalꢀrelayꢀresiduesꢀbyꢀprotonꢀmanagementꢀrescuesꢀ
proteinꢀelectronꢀhoppingꢀ
ꢀ
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4ꢀ
5ꢀ
†
†,§
†,*ꢀ
EstellaꢀF.ꢀYee ,ꢀBorisꢀDzikovski ,ꢀBrianꢀR.ꢀCrane
ꢀ
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ꢀ
ꢀ
ꢀ
ꢀ
†
DepartmentꢀofꢀChemistryꢀandꢀChemicalꢀBiology,ꢀCornellꢀUniversity,ꢀIthaca,ꢀNewꢀYorkꢀ14853,ꢀ
UnitedꢀStatesꢀꢀ
§
NationalꢀBiomedicalꢀCenterꢀforꢀAdvancedꢀESRꢀTechnologiesꢀ(ACERT),ꢀCornellꢀUniversity,ꢀIthaca,ꢀ
10ꢀ NewꢀYorkꢀ14850,ꢀUnitedꢀStatesꢀ
11ꢀ
ꢀ
1
1
2ꢀ KEYWORDS:ꢀElectronꢀtransfer,ꢀtyrosylꢀradical,ꢀcytochromeꢀcꢀperoxidase,ꢀprotonꢀtransfer,ꢀPCET,ꢀ
3ꢀ photosystemꢀII,ꢀribonucleotideꢀreductase,ꢀcompoundꢀIꢀ
ꢀ
1ꢀ
ꢀ
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4ꢀ ABSTRACT:ꢀTransientꢀtyrosineꢀandꢀtryptophanꢀradicalsꢀplayꢀkeyꢀrolesꢀinꢀtheꢀelectronꢀtransferꢀ
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5ꢀ (ET)ꢀreactionsꢀofꢀphotosystemꢀ(PS)ꢀII,ꢀribonucleotideꢀreductaseꢀ(RNR),ꢀphotolyase,ꢀandꢀmanyꢀ
16ꢀ otherꢀproteins.ꢀHowever,ꢀTyrꢀandꢀTrpꢀareꢀnotꢀfunctionallyꢀinterchangeableꢀandꢀtheꢀfactorsꢀ
17ꢀ controllingꢀtheirꢀreactivityꢀareꢀoftenꢀunclear.ꢀCytochromeꢀcꢀperoxidaseꢀ(CcP)ꢀemploysꢀaꢀTrp191 ꢀ
+
•
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8ꢀ radicalꢀtoꢀoxidizeꢀreducedꢀcytochromeꢀcꢀ(Cc).ꢀAlthoughꢀaꢀTyr191ꢀreplacementꢀalsoꢀformsꢀaꢀ
9ꢀ stableꢀradical,ꢀitꢀdoesꢀnotꢀsupportꢀrapidꢀETꢀfromꢀCc.ꢀHereꢀweꢀprobeꢀtheꢀredoxꢀpropertiesꢀofꢀCcPꢀ
0ꢀ Y191ꢀbyꢀnon-naturalꢀaminoꢀacidꢀsubstitution,ꢀalteringꢀtheꢀETꢀdrivingꢀforce,ꢀandꢀmanipulatingꢀtheꢀ
1ꢀ proticꢀenvironmentꢀofꢀY191.ꢀHigherꢀpotentialꢀfluorotyrosineꢀresiduesꢀincreaseꢀETꢀratesꢀ
•
22ꢀ marginally,ꢀbutꢀonlyꢀadditionꢀofꢀaꢀhydrogenꢀbondꢀdonorꢀtoꢀTyr191 ꢀ(viaꢀLeu232HisꢀorꢀGlu)ꢀ
2
2
2
2
3ꢀ substantiallyꢀaltersꢀactivityꢀbyꢀincreasingꢀtheꢀETꢀrateꢀbyꢀnearlyꢀ30-fold.ꢀESRꢀandꢀESEEMꢀ
4ꢀ spectroscopies,ꢀcrystallography,ꢀandꢀpH-dependentꢀETꢀkineticsꢀprovideꢀstrongꢀevidenceꢀforꢀ
•
5ꢀ hydrogenꢀbondꢀformationꢀtoꢀY191 ꢀbyꢀHis232/Glu232.ꢀRateꢀmeasurementsꢀandꢀrapidꢀfreezeꢀ
6ꢀ quenchꢀESRꢀspectroscopyꢀfurtherꢀrevealꢀdifferencesꢀinꢀradicalꢀpropagationꢀandꢀCcꢀoxidationꢀthatꢀ
•
2
7ꢀ supportꢀanꢀincreasedꢀY191 ꢀformalꢀpotentialꢀofꢀ∼200ꢀmVꢀinꢀtheꢀpresenceꢀofꢀE232.ꢀHence,ꢀY191ꢀ
•+
2
2
3
8ꢀ inactivityꢀresultsꢀfromꢀaꢀpotentialꢀdropꢀowingꢀtoꢀY191 ꢀdeprotonation.ꢀIncorporationꢀofꢀaꢀwell-
•
9ꢀ positionedꢀbaseꢀtoꢀacceptꢀandꢀdonateꢀbackꢀaꢀhydrogenꢀbondꢀupshiftsꢀtheꢀTyr ꢀpotentialꢀintoꢀaꢀ
Z D
0ꢀ rangeꢀwhereꢀitꢀcanꢀeffectivelyꢀoxidizeꢀCc.ꢀTheseꢀfindingsꢀhaveꢀimplicationsꢀforꢀtheꢀY /Y ꢀradicalsꢀ
31ꢀ ofꢀPSꢀII,ꢀhole-hoppingꢀinꢀRNRꢀandꢀcryptochrome,ꢀandꢀengineeringꢀproteinsꢀforꢀlong-rangeꢀETꢀ
3
3
2ꢀ reactions.ꢀ
3ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
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4ꢀ Introductionꢀ
5ꢀ Tryptophanꢀ(Trp)ꢀandꢀtyrosineꢀ(Tyr)ꢀradicalsꢀareꢀincreasinglyꢀrecognizedꢀasꢀessentialꢀfeaturesꢀofꢀ
1
–7
36ꢀ enzymaticꢀcatalysisꢀandꢀproteinꢀfunction .ꢀEnzymesꢀandꢀlightꢀsensorsꢀsuchꢀasꢀphotosystemꢀIIꢀ
37ꢀ (PSꢀII),ꢀribonucleotideꢀreductaseꢀ(RNR),ꢀprostaglandinꢀsynthase,ꢀphotolyases/cryptochromesꢀ
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8ꢀ andꢀBLUFꢀproteins,ꢀallꢀinvolveꢀTrpꢀandꢀTyrꢀradicalsꢀinꢀlong-rangeꢀelectronꢀtransferꢀ(ET) .ꢀTrpꢀ
1
5
9ꢀ andꢀTyrꢀresiduesꢀhaveꢀsimilarꢀformalꢀpotentialsꢀ(∼–0.9ꢀtoꢀ–1.1ꢀVꢀatꢀphysiologicalꢀpH )ꢀthatꢀareꢀ
4
0ꢀ inꢀrangeꢀofꢀtheꢀreactiveꢀsitesꢀgeneratedꢀatꢀoxo-metallocentersꢀorꢀphotoexcitedꢀpigmentsꢀinꢀ
3
,16,17
4
1ꢀ proteins.ꢀHowever,ꢀTrpꢀandꢀTyrꢀareꢀnotꢀfunctionallyꢀinterchangeable
2ꢀ differentꢀpK ꢀvaluesꢀofꢀtheirꢀrespectiveꢀradicals.ꢀHence,ꢀtheꢀproticꢀenvironmentꢀhasꢀaꢀlargeꢀ
3ꢀ effectꢀonꢀtheirꢀreactivity,ꢀandꢀproton-coupledꢀelectronꢀtransferꢀ(PCET)ꢀinꢀitsꢀvariousꢀforms
4ꢀ canꢀinfluenceꢀtheseꢀreactions.ꢀSmall-moleculeꢀexperimentsꢀhaveꢀdemonstratedꢀtheꢀimpactꢀofꢀ
ꢀowingꢀtoꢀtheꢀveryꢀ
4
a
3,7,18–21
4
ꢀ
4
2
2–35
45ꢀ surroundingꢀacidsꢀandꢀbasesꢀonꢀtheꢀPCETꢀreactionsꢀofꢀindoleꢀandꢀphenolꢀmoieties
.ꢀTheseꢀ
4
4
4
4
5
Z
6ꢀ studies,ꢀinspiredꢀbyꢀeffortsꢀtoꢀmimicꢀtheꢀredoxꢀactiveꢀtyrosineꢀresidueꢀ(Y )ꢀofꢀPSꢀII,ꢀhighlightꢀaꢀ
7ꢀ rangeꢀofꢀmechanismsꢀthatꢀareꢀdistinguishedꢀbyꢀsingleꢀversusꢀmulti-siteꢀPCET,ꢀelectron-transferꢀ
8ꢀ drivingꢀforce,ꢀsolventꢀenvironment,ꢀtheꢀpresenceꢀofꢀhydrogenꢀbondꢀrelays,ꢀandꢀproximityꢀofꢀtheꢀ
3
6
9ꢀ protonꢀdonorꢀandꢀacceptorꢀ(reviewedꢀbyꢀPannwitzꢀandꢀWenger ).ꢀYet,ꢀaꢀproteinꢀmatrixꢀ
0ꢀ providesꢀaꢀheterogeneousꢀenvironmentꢀforꢀTrpꢀandꢀTyrꢀredoxꢀchemistry,ꢀandꢀitꢀisꢀunclearꢀ
51ꢀ whetherꢀlessonsꢀlearnedꢀfromꢀmodelꢀstudies,ꢀwhichꢀareꢀoftenꢀcarriedꢀoutꢀinꢀnon-aqueousꢀ
3
5
5
2ꢀ solventsꢀ(withꢀexception ),ꢀareꢀfullyꢀapplicableꢀtoꢀtheseꢀmoreꢀcomplexꢀsystems.ꢀMoreover,ꢀ
3ꢀ mostꢀmodelꢀsystemsꢀareꢀoxidativeꢀinꢀnatureꢀandꢀdrivenꢀphotochemically,ꢀwhereasꢀproteinꢀPCETꢀ
4ꢀ canꢀbeꢀbothꢀreductiveꢀandꢀoxidativeꢀandꢀinitiatedꢀbyꢀaꢀbroadꢀclassꢀofꢀreactiveꢀgroundꢀandꢀ
5ꢀ excited-stateꢀspecies.ꢀToꢀthisꢀend,ꢀengineeredꢀproteinꢀmaquettesꢀandꢀpeptideꢀmodelsꢀhaveꢀ
6ꢀ beenꢀemployedꢀtoꢀprovideꢀaꢀmoreꢀcontrolledꢀproteinꢀenvironmentꢀforꢀprobingꢀlocalꢀeffectsꢀonꢀ
5
5
5
5
3
7–41
57ꢀ TrpꢀandꢀTyrꢀreactivity
.ꢀꢀInꢀaddition,ꢀstudiesꢀofꢀenzymesꢀsuchꢀasꢀPSꢀII,ꢀwhereinꢀtheꢀY
z
ꢀradicalꢀ
4
2,43
5
5
6
6
8ꢀ couplesꢀphotoexcitationꢀtoꢀwaterꢀsplitting
,ꢀandꢀRNR,ꢀwhereꢀincorporationꢀofꢀnon-naturalꢀTyrꢀ
4
4,45
9ꢀ analogsꢀhaveꢀallowedꢀforꢀcharacterizationꢀofꢀtrappedꢀradicals
,ꢀunderscoreꢀtheꢀcapacityꢀofꢀ
0ꢀ localꢀresiduesꢀthatꢀprovideꢀhydrogenꢀbondsꢀorꢀinfluenceꢀsolvationꢀtoꢀalterꢀtheꢀreactivityꢀofꢀTrpꢀ
1ꢀ andꢀTyrꢀresidues.ꢀInꢀsuchꢀproteinꢀsystems,ꢀmajorꢀquestionsꢀcenterꢀonꢀunderstandingꢀtheꢀ
62ꢀ specificityꢀofꢀwhichꢀTyr/TrpꢀresiduesꢀparticipateꢀinꢀETꢀreactions,ꢀhowꢀtheꢀproteinꢀmatrixꢀcontrolsꢀ
3ꢀ
ꢀ
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3ꢀ PCETꢀbyꢀalteringꢀlocalꢀformalꢀpotentialsꢀandꢀpK
a
ꢀvalues,ꢀandꢀimportantly,ꢀtheꢀroleꢀofꢀ
4
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,46–48
4ꢀ conformationalꢀchangeꢀandꢀdynamicsꢀinꢀmodulatingꢀreactivity
.ꢀAllꢀofꢀtheseꢀfactorsꢀcanꢀ
65ꢀ combineꢀtoꢀengenderꢀmulti-stepꢀorꢀhole-hoppingꢀETꢀreactionsꢀcapableꢀofꢀdirectingꢀchargeꢀ
1
4
66ꢀ propagationꢀoverꢀlongꢀdistancesꢀandꢀpreventingꢀnon-productiveꢀrecombination .ꢀ
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7ꢀ
ꢀ
6
8ꢀ Weꢀhaveꢀsoughtꢀaꢀtractable,ꢀyetꢀrelativelyꢀcomplexꢀproteinꢀsystemꢀtoꢀserveꢀasꢀaꢀmodelꢀforꢀ
9ꢀ understandingꢀmulti-stepꢀETꢀreactionsꢀthatꢀrelyꢀonꢀTrpꢀandꢀTyr.ꢀCytochromeꢀcꢀperoxidaseꢀ(CcP)ꢀ
0ꢀ andꢀcytochromeꢀcꢀ(Cc)ꢀfromꢀyeastꢀareꢀaꢀlong-studiedꢀpairꢀofꢀredoxꢀpartnersꢀthatꢀfunctionꢀtoꢀ
6
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4
9–51
71ꢀ reduceꢀperoxideꢀtoꢀwaterꢀthroughꢀinvolvementꢀofꢀaꢀTrpꢀradical
ꢀ(Schemeꢀ1).ꢀPeroxideꢀreactsꢀ
7
7
7
7
7
7
2ꢀ withꢀtheꢀCcPꢀhemeꢀtoꢀformꢀaꢀCompoundꢀIꢀ(CpdꢀI)ꢀspeciesꢀthatꢀisꢀthenꢀreducedꢀsequentiallyꢀbyꢀ
2
+
3ꢀ twoꢀmoleculesꢀofꢀCc(Fe ).ꢀCpdꢀIꢀcontainsꢀaꢀTrp191ꢀradical,ꢀwhichꢀoxidizesꢀtheꢀferrousꢀCcꢀhemeꢀ
2+
4ꢀ (Cc(Fe ))ꢀsomeꢀ20ꢀÅꢀaway.ꢀThus,ꢀtoꢀfullyꢀreduceꢀperoxideꢀeachꢀCpdꢀIꢀmustꢀreactꢀwithꢀtwoꢀCcꢀ
2
5ꢀ molecules.ꢀTheꢀfirstꢀreductionꢀofꢀCpdꢀIꢀoccursꢀwithꢀaꢀsecondꢀorderꢀrateꢀconstantꢀofꢀ2ꢀxꢀ10 ꢀtoꢀ3ꢀxꢀ
3
-1
-1
6ꢀ 10 ꢀµM ꢀsec ꢀdependingꢀonꢀconditions,ꢀwhereasꢀtheꢀsecondꢀreductionꢀofꢀCpdꢀIIꢀhasꢀaꢀrateꢀ
2
-1
-1
52,53
7ꢀ constantꢀofꢀ10ꢀtoꢀ10 ꢀµM ꢀsec ꢀfromꢀtheꢀsameꢀsite
.ꢀTheꢀsecondꢀreductionꢀisꢀslowerꢀthanꢀtheꢀ
78ꢀ firstꢀowingꢀtoꢀtheꢀequilibriumꢀofꢀstepꢀIIIꢀinꢀSchemeꢀI,ꢀwhichꢀliesꢀtoꢀtheꢀleft.ꢀꢀ
7
8
8
8
9ꢀ
ꢀ
0ꢀ Extensiveꢀstudiesꢀhaveꢀrevealedꢀaꢀcomplexꢀdependenceꢀofꢀtheꢀreactionꢀonꢀsolutionꢀconditionsꢀ
1ꢀ (e.g.,ꢀpH,ꢀionicꢀstrength,ꢀandꢀtemperature).ꢀInitialꢀencountersꢀofꢀCcꢀwithꢀCcPꢀareꢀlargelyꢀ
2ꢀ governedꢀbyꢀelectrostaticsꢀandꢀasꢀaꢀresult,ꢀtheꢀreactionꢀmechanismꢀchangesꢀconsiderablyꢀasꢀaꢀ
83ꢀ functionꢀofꢀionicꢀstrength.ꢀAlternativeꢀdockingꢀconfigurationsꢀbecomeꢀoperativeꢀatꢀlowꢀionicꢀ
84ꢀ strengthsꢀ(<ꢀ100ꢀmM)ꢀandꢀCcꢀoff-ratesꢀdecreaseꢀsubstantially.ꢀConformationalꢀgatingꢀcoupledꢀtoꢀ
8
5ꢀ dynamicꢀdockingꢀofꢀtheꢀCcP:Ccꢀcomplexꢀalsoꢀmodulateꢀreactivityꢀinꢀaꢀmannerꢀhighlyꢀdependentꢀ
5
3–64
8
6ꢀ onꢀconditions
.ꢀꢀ
8
7ꢀ
ꢀ
8
8ꢀ Importantly,ꢀsubstitutionꢀofꢀTrp191ꢀtoꢀPheꢀdropsꢀETꢀratesꢀfromꢀCcꢀtoꢀCcPꢀbyꢀmanyꢀordersꢀofꢀ
6
5–67
89ꢀ magnitude
.ꢀSurprisingly,ꢀwhenꢀW191ꢀisꢀsubstitutedꢀtoꢀTyr,ꢀaꢀstableꢀCpdꢀIꢀspeciesꢀformsꢀwithꢀ
1
7
90ꢀ aꢀradicalꢀlocalizedꢀonꢀTyr191,ꢀbutꢀETꢀisꢀslowꢀandꢀtheꢀsystemꢀdoesꢀnotꢀfunction .ꢀToꢀinvestigateꢀ
1ꢀ whyꢀTyr191ꢀwouldꢀbeꢀinactiveꢀdespiteꢀformingꢀaꢀstableꢀradical,ꢀweꢀhaveꢀmanipulatedꢀtheꢀredoxꢀ
9
4ꢀ
ꢀ
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2ꢀ andꢀprotonꢀaffinityꢀofꢀtheꢀTyrꢀsiteꢀbyꢀintroducingꢀfluorotyrosineꢀderivativesꢀ(FTyr)ꢀandꢀhydrogen-
3ꢀ bondingꢀpartnersꢀforꢀtheꢀphenolicꢀhydroxylꢀmoiety.ꢀTheseꢀexperimentsꢀareꢀcarriedꢀoutꢀunderꢀ
94ꢀ conditionsꢀwhereinꢀproteinꢀexchangeꢀandꢀreactionꢀwithꢀperoxideꢀareꢀrelativelyꢀrapidꢀcomparedꢀ
95ꢀ toꢀETꢀfromꢀCcꢀtoꢀtheꢀCcPꢀW191ꢀvariants.ꢀTheꢀFTyrsꢀincreaseꢀETꢀrates,ꢀbutꢀnotꢀasꢀeffectivelyꢀasꢀ
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6ꢀ additionꢀofꢀaꢀconjugateꢀbaseꢀthatꢀhydrogenꢀbondsꢀtoꢀTyr.ꢀThroughꢀaꢀseriesꢀofꢀspectroscopic,ꢀ
7ꢀ structural,ꢀandꢀkineticꢀstudies,ꢀweꢀsurmiseꢀthatꢀtheꢀneutralꢀTyrꢀradicalꢀisꢀtooꢀlowꢀinꢀpotentialꢀtoꢀ
8ꢀ rapidlyꢀoxidizeꢀCc,ꢀbutꢀadditionꢀofꢀaꢀstrongꢀhydrogenꢀbondꢀtoꢀtheꢀTyrꢀradicalꢀupshiftsꢀitsꢀ
2+
9
9ꢀ potentialꢀ∼200ꢀmV,ꢀwhichꢀisꢀsufficientꢀtoꢀaccelerateꢀlong-rangeꢀETꢀfromꢀCc(Fe ).ꢀAlthoughꢀtheꢀ
1
1
1
00ꢀ activeꢀTyrꢀradicalꢀisꢀnotꢀconvertedꢀtoꢀaꢀcation,ꢀwhichꢀhasꢀonlyꢀbeenꢀobservedꢀforꢀTyrꢀonꢀveryꢀfastꢀ
68
01ꢀ timeꢀscalesꢀinꢀbiologicalꢀsystems ,ꢀtheꢀeffectꢀofꢀaꢀsuitablyꢀpositionedꢀconjugateꢀbaseꢀonꢀ
z
02ꢀ reactivityꢀisꢀquiteꢀdramatic.ꢀTheꢀCcP:CcꢀTyrꢀradicalꢀisꢀanalogousꢀꢀtoꢀtheꢀY ꢀradicalꢀofꢀPSꢀII,ꢀwhichꢀ
103ꢀ requiresꢀinteractionꢀwithꢀaꢀconservedꢀHisꢀresidueꢀtoꢀtransitꢀelectronsꢀbetweenꢀtheꢀP680ꢀspecialꢀ
6
9–71
1
1
1
1
1
04ꢀ chlorophyllꢀdimersꢀandꢀtheꢀMn-clusterꢀofꢀtheꢀoxygen-evolvingꢀcomplex
.ꢀUnlikeꢀPSꢀII,ꢀtheꢀ
05ꢀ CcP:Ccꢀsystemꢀisꢀreadilyꢀstudiedꢀbyꢀcrystallographyꢀandꢀamenableꢀtoꢀincorporationꢀofꢀnon-
06ꢀ naturalꢀresidues,ꢀandꢀunlikeꢀRNR,ꢀinꢀCcP:Cc,ꢀtheꢀintermediateꢀTyrꢀradicalꢀisꢀstableꢀandꢀdoesꢀnotꢀ
07ꢀ requireꢀmodificationꢀtoꢀbeꢀstudied.ꢀThus,ꢀCcP:Ccꢀprovidesꢀanꢀexcellentꢀmodelꢀforꢀunderstandingꢀ
08ꢀ howꢀenergy-transducingꢀproteinsꢀcontrolꢀlong-rangeꢀETꢀreactionsꢀthroughꢀredox-activeꢀ
109ꢀ residues.ꢀꢀ
1
10ꢀ
ꢀ
111ꢀ Schemeꢀ1ꢀ–ꢀReactionsꢀofꢀWTꢀCcP:Cc.ꢀProtonationꢀofꢀtheꢀferricꢀhydroxideꢀinꢀstepꢀIIIꢀmayꢀlagꢀorꢀ
•
+
1
1
1
1
12ꢀ precedeꢀreductionꢀofꢀW191 ꢀinꢀstepꢀIVꢀdependingꢀonꢀconditions.ꢀ
13ꢀ
ꢀ
14ꢀ Resultsꢀ
15ꢀ ElectronꢀtransferꢀratesꢀofꢀY191ꢀvariantsꢀ
1
7
116ꢀ GivenꢀthatꢀtheꢀstructureꢀCcPꢀW191YꢀisꢀnearlyꢀidenticalꢀtoꢀWT ,ꢀtheꢀinactivityꢀofꢀCpdꢀIꢀmostꢀlikelyꢀ
117ꢀ derivesꢀfromꢀchangesꢀinꢀtheꢀformalꢀpotentialꢀand/orꢀprotonꢀaffinityꢀofꢀtheꢀradical.ꢀToꢀalterꢀtheseꢀ
1
18ꢀ propertiesꢀofꢀY191,ꢀweꢀincorporatedꢀnon-naturalꢀfluorotyrosineꢀresiduesꢀinꢀplaceꢀofꢀY191,ꢀ
5ꢀ
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19ꢀ modifiedꢀitsꢀproticꢀenvironmentꢀbyꢀengineeringꢀbasic,ꢀaddedꢀhydrogenꢀbondingꢀresiduesꢀ
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20ꢀ proximalꢀtoꢀtheꢀphenoxylꢀmoiety,ꢀandꢀshiftedꢀtheꢀformalꢀpotentialꢀofꢀtheꢀCc(Fe )ꢀdonorꢀthroughꢀ
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121ꢀ residueꢀsubstitutionsꢀofꢀknownꢀeffect .ꢀTheꢀ2,3,5-trifluorotyrosineꢀandꢀ3,5-difluorotyrosineꢀ
122ꢀ aminoꢀacidsꢀwereꢀsynthesizedꢀbyꢀtyrosineꢀphenolꢀlyase,ꢀpurified,ꢀandꢀincorporatedꢀintoꢀCcPꢀ
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23ꢀ usingꢀanꢀE3ꢀaminoacyl-tRNAꢀsynthetase/tRNAꢀamberꢀsuppressionꢀsystem
.ꢀTheꢀuseꢀofꢀ
24ꢀ fluorotyrosineꢀresiduesꢀtoꢀmanipulateꢀE°’ꢀandꢀpK
a
ꢀhasꢀbeenꢀwellꢀestablishedꢀbyꢀpreviousꢀstudiesꢀ
44,75–78
25ꢀ ofꢀPCETꢀmechanismsꢀandꢀpathwaysꢀinꢀPSꢀII,ꢀRNR,ꢀandꢀBLUF-domains
.ꢀTheꢀparticularꢀ
ꢀperturbationsꢀatꢀ
26ꢀ analoguesꢀthatꢀwereꢀselectedꢀprovideꢀrelativelyꢀsmallꢀformalꢀpotentialꢀandꢀpK
a
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a
27ꢀ theꢀ191ꢀsiteꢀ(approximatelyꢀ±ꢀ50ꢀmVꢀandꢀ−∼3.5ꢀpK ꢀunits) .ꢀAlso,ꢀunderꢀtheꢀpHꢀ6ꢀconditionsꢀofꢀ
1
28ꢀ theꢀexperiments,ꢀtheseꢀresiduesꢀ(pK
a 3 2
ꢀ=ꢀ6.4ꢀforꢀ2,3,5-F Yꢀandꢀ7.2ꢀforꢀ3,5-F Y)ꢀremainꢀprimarilyꢀ
1
29ꢀ protonatedꢀpriorꢀtoꢀperoxideꢀreaction.ꢀYet,ꢀtyrosylꢀradicalsꢀgreatlyꢀfavorꢀtheꢀneutralꢀ
1
5,39
130ꢀ deprotonatedꢀformꢀowingꢀtoꢀaꢀlowꢀintrinsicꢀpK
a
ꢀofꢀ−2ꢀ
,ꢀalthoughꢀaꢀproteinꢀmatrixꢀcanꢀalterꢀ
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9,80
•+
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31ꢀ thisꢀvalue
32ꢀ toꢀinteractionꢀwithꢀAsp235
33ꢀ acceptor,ꢀsuchꢀasꢀHis,ꢀAsp,ꢀorꢀGlu,ꢀcouldꢀpotentiallyꢀstabilizeꢀtheꢀcationicꢀformꢀorꢀfacilitateꢀ
.ꢀNotably,ꢀtheꢀWTꢀW191 ꢀradicalꢀmaintainsꢀaꢀcationicꢀstateꢀinꢀCpdꢀI,ꢀowingꢀinꢀpartꢀ
8
1–83
.ꢀHence,ꢀcoordinationꢀofꢀtyrosylꢀradicalsꢀtoꢀaꢀnearbyꢀprotonꢀ
3
9,84
34ꢀ protonꢀtransferꢀeventsꢀcoupledꢀtoꢀET
.ꢀToꢀmanipulateꢀtheꢀproticꢀenvironmentꢀofꢀY191,ꢀ
35ꢀ neighboringꢀLeu232ꢀwasꢀreplacedꢀwithꢀeitherꢀHisꢀorꢀGluꢀ(W191Y:L232HꢀandꢀW191Y:L232E,ꢀ
2
+
136ꢀ respectively).ꢀCorrespondingly,ꢀweꢀalsoꢀalteredꢀtheꢀformalꢀpotentialꢀofꢀtheꢀCc(Fe )ꢀdonorꢀbyꢀ
7
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37ꢀ replacingꢀTyr48ꢀwithꢀLysꢀtoꢀupshiftꢀtheꢀpotentialꢀ120ꢀmV .ꢀꢀ
38ꢀ
ꢀ
2+
39ꢀ Cc(Fe )ꢀoxidationꢀratesꢀinꢀsingleꢀandꢀmultipleꢀturnoverꢀconditionsꢀdependꢀonꢀdrivingꢀforceꢀ
40ꢀ andꢀaꢀconjugateꢀbaseꢀ
2
+
1
41ꢀ ETꢀratesꢀfromꢀCc(Fe )ꢀtoꢀtheꢀCpdꢀI-likeꢀspeciesꢀofꢀtheꢀCcPꢀvariantsꢀwereꢀmeasuredꢀinꢀsingleꢀ
2
+
2 2
142ꢀ turnoverꢀ(1ꢀµMꢀCcP,ꢀ2ꢀµMꢀH O ,ꢀ2ꢀµMꢀCc(Fe ))ꢀandꢀmultipleꢀturnoverꢀformatsꢀꢀ(1ꢀµMꢀCcP,ꢀ10ꢀ
2
+
143ꢀ µMꢀH
2
O
2
,ꢀ30ꢀµMꢀCc(Fe ))ꢀ(Figureꢀ1).ꢀIonicꢀstrengthꢀconditionsꢀandꢀpHꢀwereꢀchosenꢀtoꢀminimizeꢀ
1
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44ꢀ anyꢀsecond-siteꢀbindingꢀofꢀCcꢀtoꢀCcP.ꢀProteinꢀconcentrationsꢀwereꢀasꢀhighꢀasꢀpracticalꢀtoꢀprovideꢀ
45ꢀ goodꢀspectralꢀproperties.ꢀUnderꢀtheꢀ100ꢀmMꢀpotassiumꢀphosphateꢀ(KPi)ꢀbufferꢀconditionsꢀ(ionicꢀ
-1 52,53
1
46ꢀ strengthꢀ∼110ꢀmMꢀatꢀpHꢀ6),ꢀCcꢀoff-ratesꢀareꢀ>ꢀ2000ꢀs ꢀ
ꢀandꢀperoxideꢀreactsꢀwithꢀCcPꢀtoꢀ
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47ꢀ produceꢀCpdꢀIꢀwithꢀaꢀsecond-orderꢀrateꢀconstantꢀofꢀ∼4ꢀxꢀ10 M ꢀs ꢀ(orꢀaꢀpseudo-first-orderꢀrateꢀ
-
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48ꢀ constantꢀofꢀ400ꢀs ꢀatꢀ10ꢀµMꢀH
2
O
2
ꢀ ).ꢀTheꢀdissociationꢀconstantꢀ(K
D
)ꢀofꢀCcꢀforꢀCcPꢀunderꢀtheseꢀ
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49ꢀ conditionsꢀisꢀ∼8ꢀµM ,ꢀwhichꢀimpliesꢀthatꢀ>ꢀ80%ꢀofꢀCcP:Ccꢀcomplexꢀisꢀassociatedꢀinꢀtheꢀmultipleꢀ
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150ꢀ turnoverꢀcase,ꢀbutꢀlittleꢀcomplexꢀisꢀassociatedꢀinꢀtheꢀsingleꢀturnoverꢀcase.ꢀꢀAꢀsecond-orderꢀrateꢀ
-
1 -1 86
1
M
51ꢀ constantꢀderivedꢀfromꢀsteadyꢀstateꢀanalysisꢀ(kcat/K ꢀ∼200ꢀµM ꢀs ꢀ )ꢀmatchesꢀwellꢀwithꢀtheꢀ
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152ꢀ second-orderꢀrateꢀconstantꢀforꢀtheꢀslowerꢀsecondꢀreductionꢀofꢀCpdꢀIIꢀbyꢀCc ꢀ(stepꢀIVꢀinꢀSchemeꢀ
1
53ꢀ I),ꢀwhichꢀplacesꢀanꢀupperꢀboundꢀbelowꢀwhichꢀETꢀtoꢀtheꢀmodifiedꢀ191ꢀsiteꢀwillꢀbeꢀrate-limiting.ꢀꢀ
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ꢀ
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55ꢀ
56ꢀ
57ꢀ
58ꢀ
59ꢀ
Figureꢀ1ꢀ—ꢀCcꢀoxidationꢀreactionsꢀunderꢀmultipleꢀturnoverꢀconditions.ꢀ(A)ꢀOxidationꢀofꢀCc(Fe )ꢀbyꢀCcPꢀ
variantsꢀwasꢀmeasuredꢀatꢀAbs550ꢀnm−Abs540ꢀnm,ꢀwithꢀvaluesꢀnormalizedꢀfromꢀ0ꢀtoꢀ1ꢀ(100ꢀmMꢀpotassiumꢀ
phosphateꢀ(KPi),ꢀpHꢀ6ꢀforꢀallꢀvariantsꢀexceptꢀforꢀW191Y:L232H,ꢀwhichꢀwasꢀatꢀpHꢀ7).ꢀ(B)ꢀProgressꢀcurvesꢀatꢀ
4
+
Abs434ꢀnm,ꢀreflectꢀformationꢀandꢀdecayꢀofꢀtheꢀferrylꢀ(Fe =O)ꢀspecies.ꢀ(Curvesꢀwereꢀnormalizedꢀandꢀshiftedꢀ
vertically).ꢀꢀ
ꢀ
160ꢀ
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61ꢀ OurꢀrateꢀmeasurementsꢀrelyꢀonꢀmonitoringꢀtheꢀlossꢀofꢀCc(Fe )ꢀandꢀformation/decayꢀofꢀtheꢀ
4+
2+
62ꢀ ferrylꢀ(Fe =O)ꢀCcPꢀuponꢀperoxideꢀaddition.ꢀNotably,ꢀETꢀbetweenꢀCc(Fe )ꢀandꢀtheꢀ191ꢀradicalꢀ
4+
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63ꢀ (Schemeꢀ1)ꢀisꢀexpectedꢀtoꢀbeꢀslowꢀcomparedꢀtoꢀoxidationꢀofꢀtheꢀ191ꢀresidueꢀbyꢀFe =OꢀCcP .ꢀInꢀ
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+
64ꢀ theꢀmultipleꢀturnoverꢀscenario,ꢀETꢀfromꢀCc(Fe )ꢀandꢀregenerationꢀofꢀCpdꢀIꢀwithꢀperoxideꢀ
2
+
165ꢀ producesꢀaꢀlowꢀlevelꢀofꢀtheꢀferrylꢀspeciesꢀthatꢀincreasesꢀasꢀtheꢀconcentrationꢀofꢀCc(Fe )ꢀdrops,ꢀ
1
66ꢀ onlyꢀtoꢀthenꢀsubsideꢀwhenꢀperoxideꢀisꢀexpendedꢀ(Figureꢀ1B).ꢀCorrelationꢀofꢀtheꢀrateꢀconstantsꢀ
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67ꢀ fromꢀtheꢀsingleꢀturnoverꢀandꢀmultipleꢀturnoverꢀregimesꢀindicatesꢀthatꢀunderꢀtheseꢀconditions,ꢀ
68ꢀ proteinꢀexchangeꢀkineticsꢀdoꢀnotꢀdominateꢀtheꢀratesꢀofꢀCcꢀoxidationꢀ(Figureꢀ2).ꢀNonetheless,ꢀforꢀ
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169ꢀ variantsꢀofꢀlowꢀreactivity,ꢀtheꢀmultipleꢀturnoverꢀratesꢀlagꢀcomparedꢀtoꢀthoseꢀofꢀtheꢀsingleꢀ
170ꢀ turnoverꢀrates,ꢀtherebyꢀindicatingꢀthatꢀatꢀslowꢀratesꢀofꢀET,ꢀproteinꢀexchangeꢀdoesꢀcompeteꢀtoꢀ
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71ꢀ someꢀextent,ꢀprobablyꢀowingꢀtoꢀtheꢀincreasingꢀconcentrationꢀofꢀinterferingꢀCc(Fe )ꢀasꢀperoxideꢀ
72ꢀ reductionꢀproceeds.ꢀꢀGivenꢀtheꢀkineticꢀparametersꢀdescribedꢀabove,ꢀthisꢀphenomenonꢀ
73ꢀ combinedꢀwithꢀmixingꢀlimitationsꢀlikelyꢀreducesꢀtheꢀobservedꢀWTꢀrateꢀconstantꢀinꢀthisꢀformat.ꢀ
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ꢀ
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76ꢀ
77ꢀ
78ꢀ
79ꢀ
Figureꢀ2ꢀ—ꢀCorrelationꢀbetweenꢀsingleꢀandꢀmultipleꢀturnoverꢀrateꢀconstantsꢀforꢀCcꢀoxidation.ꢀ(A)ꢀSingleꢀ
turnoverꢀrateꢀconstantsꢀobtainedꢀinꢀ100ꢀmMꢀKPiꢀbuffer,ꢀpHꢀ6ꢀ(SupplementalꢀTableꢀ1).ꢀ(B)ꢀMultipleꢀ
turnoverꢀrateꢀconstantsꢀinꢀ100ꢀmMꢀKPiꢀbuffer,ꢀpHꢀ6ꢀ(SupplementalꢀTableꢀ1).ꢀ(C)ꢀCorrelationꢀbetweenꢀ
singleꢀandꢀmultipleꢀturnoverꢀrateꢀconstantsꢀindicatesꢀthatꢀCcꢀoxidationꢀisꢀnotꢀdominatedꢀbyꢀproteinꢀ
exchangeꢀkineticsꢀinꢀtheꢀmultipleꢀturnoverꢀformat.ꢀ
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80ꢀ
ꢀ
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81ꢀ WTꢀCcPꢀrapidlyꢀoxidizesꢀCc(Fe )ꢀandꢀaꢀtransientꢀferrylꢀsignalꢀ(Abs434ꢀnm)ꢀdevelopsꢀthatꢀthenꢀ
2
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182ꢀ dissipatesꢀoverꢀtheꢀcourseꢀofꢀtheꢀpseudo-first-orderꢀdecayꢀasꢀtheꢀpoolꢀofꢀCc(Fe )ꢀdepletesꢀ
2
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183ꢀ (Figureꢀ1B).ꢀInꢀcontrast,ꢀW191YꢀCcPꢀreactsꢀslowlyꢀwithꢀCc(Fe )ꢀandꢀhasꢀsimilarꢀinactivityꢀasꢀ
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84ꢀ W191Gꢀ(Figureꢀ2),ꢀinꢀwhichꢀaꢀsolvent-filledꢀcavityꢀreplacesꢀtheꢀradicalꢀsite .ꢀAsꢀaꢀresult,ꢀtheꢀCcPꢀ
85ꢀ ferrylꢀpersistsꢀforꢀminutesꢀ(Figureꢀ1B)ꢀandꢀonlyꢀcompletelyꢀreducesꢀtoꢀtheꢀferricꢀstateꢀduringꢀtheꢀ
65
86ꢀ slowꢀphaseꢀofꢀrecovery .ꢀReplacementꢀofꢀTyr191ꢀwithꢀaꢀstrongerꢀoxidantꢀ(2,3,5-
87ꢀ trifluorotyrosine)ꢀmarginallyꢀincreasesꢀtheꢀETꢀrateꢀbyꢀhalfꢀrelativeꢀtoꢀW191Y,ꢀconsistentꢀwithꢀtheꢀ
•
• 44,75
188ꢀ predictedꢀformalꢀpotentialꢀincreaseꢀofꢀtheꢀFTyr ꢀ(E’°ꢀ≅ꢀ+40ꢀmVꢀrelativeꢀtoꢀTyr )
.ꢀComparably,ꢀ
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89ꢀ replacementꢀofꢀresidueꢀ191ꢀwithꢀaꢀlowerꢀformalꢀpotentialꢀ3,5-difluorotyrosineꢀmoietyꢀ(E’°ꢀ≅ꢀ
4
4,75
90ꢀ −25ꢀtoꢀ−50ꢀmVꢀrelativeꢀtoꢀTyr)
ꢀresultsꢀinꢀaꢀsimilarꢀCcꢀoxidationꢀrateꢀtoꢀthatꢀofꢀTyr191ꢀ(Figureꢀ
91ꢀ 2A).ꢀTheseꢀresultsꢀindicateꢀthatꢀtheꢀCcP:CcꢀETꢀratesꢀareꢀsensitiveꢀtoꢀtheꢀTyr191ꢀpotential,ꢀbutꢀaꢀ
92ꢀ modestꢀupshiftꢀinꢀpotentialꢀofꢀtheꢀY191ꢀsiteꢀdoesꢀnotꢀrescueꢀactivityꢀtoꢀanꢀappreciableꢀextent.ꢀ
93ꢀ
ꢀ
194ꢀ ExaminationꢀofꢀtheꢀCcPꢀW191YꢀcrystalꢀstructureꢀsuggestedꢀthatꢀsubstitutionsꢀofꢀLeu232ꢀtoꢀHisꢀ
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95ꢀ andꢀGluꢀwouldꢀaffectꢀtheꢀproticꢀenvironmentꢀnearꢀtheꢀY191ꢀhydroxylꢀgroup.ꢀIncorporationꢀofꢀ
96ꢀ L232HꢀintoꢀtheꢀW191YꢀvariantꢀcausesꢀlittleꢀchangeꢀtoꢀETꢀrateꢀconstantsꢀatꢀpHꢀ6,ꢀbutꢀproducesꢀaꢀ
97ꢀ nearlyꢀthree-foldꢀincreaseꢀinꢀactivityꢀatꢀpHꢀ7ꢀunderꢀmultipleꢀturnoverꢀconditionsꢀ(Figureꢀ3;ꢀ
98ꢀ SupplementalꢀTableꢀ1).ꢀThisꢀrateꢀincreaseꢀthenꢀdiminishesꢀatꢀhigherꢀpH.ꢀAtꢀoptimumꢀpH,ꢀtheꢀCcꢀ
99ꢀ oxidationꢀcurveꢀchangesꢀfromꢀbiexponentialꢀtoꢀaꢀWT-like,ꢀmonoexponentialꢀdecay,ꢀindicatingꢀ
200ꢀ thatꢀtheꢀbiexponentialꢀcharacteristicsꢀareꢀlikelyꢀdueꢀtoꢀaꢀmixedꢀpopulationꢀofꢀslowlyꢀexchangingꢀ
2
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01ꢀ conformationalꢀorꢀprotonationꢀstatesꢀ(Figureꢀ3C).ꢀCurveꢀfittingꢀofꢀtheꢀdataꢀtoꢀaꢀtwo-protonꢀ
02ꢀ ionizationꢀmodelꢀ(Equationꢀ1)ꢀindicatesꢀtwoꢀpK
a
ꢀvaluesꢀ(6.46ꢀandꢀ7.24)ꢀ–ꢀoneꢀforꢀHis232ꢀ
•
03ꢀ deprotonationꢀandꢀanotherꢀweꢀpresumeꢀtoꢀbeꢀtheꢀhydrogen-bondedꢀprotonꢀtoꢀY191 ꢀ(Figureꢀ
04ꢀ 3D).ꢀꢀ
!!"#
!!! !!"
2
05ꢀ
ꢀ
ꢀ!"# = !!!"
!ꢀ
!!"!"!!!"
(1)ꢀ
!
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06ꢀ Additionalꢀevidenceꢀforꢀrateꢀaccelerationꢀfromꢀtheꢀintroductionꢀofꢀaꢀhydrogen-bondedꢀprotonꢀ
•
07ꢀ toꢀY191 ꢀwasꢀattainedꢀbyꢀloweringꢀtheꢀpK
a
ꢀofꢀtheꢀcoordinatingꢀbaseꢀbyꢀsubstitutionꢀofꢀresidueꢀ
08ꢀ 232ꢀwithꢀGlu.ꢀRemarkably,ꢀW191Y:L232EꢀyieldsꢀsimilarꢀCcꢀoxidationꢀbehaviorꢀasꢀWTꢀCcPꢀwithꢀanꢀ
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09ꢀ ∼30ꢀ×ꢀaccelerationꢀinꢀCc(Fe )ꢀoxidationꢀrateꢀcomparedꢀtoꢀtheꢀW191Yꢀparentꢀ(Figureꢀ1,2;ꢀ
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10ꢀ SupplementalꢀTableꢀ1).ꢀInꢀW191Y:L232E,ꢀferrylꢀbuild-upꢀandꢀdecayꢀkineticsꢀapproachesꢀthoseꢀ
11ꢀ observedꢀforꢀWTꢀCcPꢀ(Figureꢀ1B).ꢀꢀTheꢀresidueꢀ232ꢀsubstitutionsꢀdoꢀnotꢀimpactꢀCpdꢀIꢀformation,ꢀ
12ꢀ asꢀassessedꢀbyꢀreactingꢀCcPꢀaloneꢀwithꢀperoxideꢀ(SupplementalꢀFigureꢀ1).ꢀTheꢀGluꢀvariantꢀalsoꢀ
13ꢀ exhibitsꢀaꢀpHꢀdependencyꢀunseenꢀinꢀW191Yꢀ(Figureꢀ3A,B).ꢀꢀAtꢀhigherꢀpHs,ꢀtheꢀCcꢀoxidationꢀrateꢀ
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214ꢀ slowsꢀtoꢀthatꢀofꢀW191Y,ꢀagainꢀsuggestingꢀthatꢀdeprotonationꢀofꢀaꢀhydrogen-bondedꢀcomplexꢀ
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15ꢀ composedꢀofꢀY191ꢀandꢀtheꢀconjugateꢀbaseꢀreducesꢀreductionꢀratesꢀ(SupplementalꢀTableꢀ1).ꢀꢀTheꢀ
16ꢀ factꢀthatꢀGlu232ꢀhasꢀaꢀlargerꢀaffectꢀonꢀrateꢀthanꢀHis,ꢀdespiteꢀhavingꢀtheꢀlowerꢀpKa,ꢀsuggestsꢀthatꢀ
17ꢀ theꢀconjugateꢀbaseꢀdoesꢀnotꢀfunctionꢀtoꢀincreaseꢀtheꢀrateꢀofꢀprotonꢀtransferꢀfromꢀY191.ꢀ
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18ꢀ
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219ꢀ
Figureꢀ3ꢀ–ꢀConjugateꢀbaseꢀnextꢀtoꢀTyr191ꢀinꢀCcPꢀaltersꢀtheꢀpHꢀdependenceꢀofꢀCcꢀoxidation.ꢀKineticꢀtracesꢀ
ofꢀCcꢀoxidationꢀ(lowerꢀcurves)ꢀandꢀCcPꢀferrylꢀformation/decayꢀ(upperꢀcurves)ꢀwereꢀobtainedꢀwithꢀeitherꢀ
(A)ꢀW191YꢀCcP,ꢀ(B)ꢀW191Y:L232EꢀCcP,ꢀorꢀ(C)ꢀW191Y:L232HꢀCcP.ꢀW191YꢀshowsꢀlittleꢀvariationꢀwithꢀpHꢀ
whereasꢀoxidationꢀratesꢀwithꢀeitherꢀtheꢀGlu232ꢀorꢀHis232ꢀvariantꢀgivesꢀstrongꢀpHꢀdependenciesꢀ
indicativeꢀofꢀaꢀhydrogenꢀbondꢀtoꢀTyr191.ꢀ(D)ꢀDependenceꢀofꢀCc(Fe )ꢀoxidationꢀratesꢀonꢀpHꢀforꢀCcPꢀ
a
W191Y:L232Hꢀinꢀ100ꢀmMꢀKPiꢀbuffers.ꢀFitꢀtoꢀaꢀtwo-protonꢀionizationꢀmodelꢀ(Equationꢀ1)ꢀgivesꢀtwoꢀpK ꢀ
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valuesꢀofꢀ6.46ꢀandꢀ7.24,ꢀwhichꢀlikelyꢀcoincideꢀwithꢀtheꢀpK ꢀofꢀHisꢀandꢀtheꢀhydrogen-bondedꢀcomplex,ꢀ
226ꢀ
227ꢀ
respectively.ꢀ
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28ꢀ Somewhatꢀsurprisingly,ꢀtheꢀeffectsꢀofꢀFTyrꢀsubstitutionꢀandꢀconjugateꢀbaseꢀadditionꢀwereꢀnotꢀ
29ꢀ synergistic.ꢀTheꢀL232EꢀsubstitutionꢀinꢀconcertꢀwithꢀtheꢀFTyrꢀvariantꢀW191-F Y,ꢀproducesꢀaꢀfastꢀ
Y,ꢀbutꢀalsoꢀ
231ꢀ exhibitsꢀaꢀmuchꢀslowerꢀsecondaryꢀphase.ꢀAlthoughꢀtheꢀoriginꢀofꢀtheꢀslowꢀphaseꢀisꢀunclearꢀitꢀmayꢀ
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230ꢀ phaseꢀofꢀCc(Fe )ꢀoxidationꢀthatꢀisꢀintermediateꢀinꢀrateꢀtoꢀW191Y:L232EꢀandꢀW191-F
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32ꢀ oweꢀtoꢀoxidativeꢀmodificationꢀofꢀtheꢀvariantꢀprotein.ꢀRegardingꢀtheꢀfastꢀphase,ꢀtheꢀlowerꢀpK ꢀofꢀ
73
33ꢀ theꢀFTyr ꢀinꢀconjunctionꢀwithꢀitsꢀelevatedꢀformalꢀpotentialꢀmayꢀcontributeꢀtoꢀaꢀradicalꢀlessꢀ
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34ꢀ readilyꢀreducedꢀthanꢀinꢀW191Y:L232E,ꢀbutꢀmoreꢀsoꢀthanꢀinꢀW191-F Yꢀaloneꢀ(Schemeꢀ1).ꢀ
35ꢀ
ꢀ
236ꢀ Proton-coupledꢀETꢀreactionsꢀoftenꢀproduceꢀsubstantialꢀdeuteriumꢀkineticꢀisotopeꢀeffectsꢀifꢀtheꢀ
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37ꢀ protonꢀandꢀelectronꢀtransfersꢀareꢀsynchronous.ꢀOvernightꢀincubationꢀofꢀW191YꢀCcP:CcꢀinꢀaꢀD
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38ꢀ basedꢀphosphateꢀbufferꢀcausesꢀonlyꢀminorꢀchangesꢀtoꢀtheꢀCc(Fe )ꢀoxidationꢀrateꢀ(k /k ꢀ≅ꢀ1.2ꢀ–ꢀ
H D
39ꢀ 1.4),ꢀbutꢀdifferencesꢀareꢀnotꢀstatisticallyꢀsignificantꢀ(pꢀ=ꢀ0.80,ꢀNꢀ=ꢀ4).ꢀInꢀcontrast,ꢀW191Y:L232Eꢀ
40ꢀ CcP:Ccꢀshowsꢀaꢀslightlyꢀlargerꢀsolventꢀisotopeꢀeffectꢀthatꢀisꢀstatisticallyꢀsignificantꢀunderꢀsingleꢀ
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H D
41ꢀ turnoverꢀconditionsꢀ(k /k ꢀ≅ꢀ1.7;ꢀkobs,ꢀD2Oꢀ=ꢀ0.09ꢀ±ꢀ0.04ꢀs ꢀversusꢀkobs,ꢀH2Oꢀ=ꢀ0.166ꢀ±ꢀ0.007ꢀs ;ꢀpꢀ=ꢀ
-
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242ꢀ 4.6ꢀ×10 ,ꢀNꢀ=ꢀ12).ꢀ
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ꢀ
44ꢀ Toꢀexploreꢀtheꢀeffectsꢀofꢀalteringꢀtheꢀpotentialꢀofꢀtheꢀdonor,ꢀweꢀproducedꢀaꢀCcꢀY48Kꢀvariant,ꢀ
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45ꢀ whichꢀisꢀknownꢀtoꢀupshiftꢀtheꢀCc(Fe )ꢀpotentialꢀfromꢀ290ꢀmVꢀtoꢀ407ꢀmV .ꢀAsꢀexpected,ꢀETꢀ
-
46ꢀ ratesꢀtoꢀW191Y:L232EꢀareꢀdecreasedꢀwithꢀCcꢀY48Kꢀ(fromꢀkobsꢀ=ꢀ0.189ꢀ±ꢀ0.006ꢀtoꢀ0.156ꢀ±ꢀ0.005ꢀs
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47ꢀ
;ꢀpꢀ=ꢀ4.44ꢀ×ꢀ10 ;ꢀNꢀ=ꢀ4ꢀandꢀ5,ꢀrespectively;ꢀSupplementalꢀTableꢀ1),ꢀagainꢀshowingꢀthatꢀtheꢀETꢀ
•
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248ꢀ rateꢀbetweenꢀY191 ꢀandꢀCc(Fe )ꢀisꢀsensitiveꢀtoꢀdrivingꢀforce.ꢀAꢀcrystalꢀstructureꢀofꢀtheꢀ
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49ꢀ CcP(W191Y):Cc(Y48K)ꢀcomplexꢀ(PDBꢀ6P43)ꢀconfirmsꢀthatꢀtheꢀsubstitutionsꢀcauseꢀnoꢀsignificantꢀ
50ꢀ structuralꢀchangesꢀtoꢀeitherꢀproteinꢀorꢀtoꢀtheirꢀassociationꢀmodeꢀ(SupplementalꢀFigureꢀ2).ꢀꢀ
51ꢀ
ꢀ
52ꢀ CrystalꢀstructuresꢀofꢀW191YꢀvariantsꢀrevealꢀhydrogenꢀbondsꢀtoꢀY191ꢀ
53ꢀ CrystalꢀstructuresꢀofꢀW191Yꢀ(PDBꢀ5CIH;ꢀFigureꢀ4A),ꢀW191Y:L232Eꢀ(PDBꢀ6P41;ꢀFigureꢀ4B),ꢀandꢀ
254ꢀ W191Y:L232Hꢀ(PDBꢀ6P42;ꢀFigureꢀ4C)ꢀallꢀinꢀcomplexꢀwithꢀCcꢀ(SupplementalꢀTableꢀ2)ꢀrevealꢀanꢀ
55ꢀ identicalꢀinterfaceꢀbetweenꢀCcPꢀandꢀCc,ꢀalongꢀwithꢀpositionsꢀofꢀtheꢀ232ꢀsideꢀchainsꢀthatꢀindicateꢀ
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56ꢀ hydrogenꢀbondsꢀtoꢀY191ꢀ(Figureꢀ4D).ꢀTheꢀactiveꢀsiteꢀofꢀtheꢀhemeꢀcenters,ꢀincludingꢀcatalyticallyꢀ
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57ꢀ essentialꢀresiduesꢀArg48,ꢀTrp51,ꢀandꢀHis52ꢀareꢀnotꢀaffectedꢀbyꢀtheꢀsubstitutions
.ꢀꢀ
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259ꢀ
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Figureꢀ4ꢀ–ꢀHydrogenꢀbondꢀenvironmentsꢀsurroundingꢀresidueꢀ191ꢀinꢀcrystalꢀstructuresꢀofꢀCcP:Ccꢀ
complexes.ꢀF ꢀ–ꢀF ꢀdifferenceꢀmapsꢀofꢀ(A)ꢀW191YꢀCcP,ꢀ(B)ꢀW191Y:L232EꢀCcP,ꢀandꢀ(C)ꢀW191Y:L232Hꢀwithꢀ
o
c
theꢀhemeꢀandꢀresiduesꢀ191ꢀandꢀ232ꢀomittedꢀfromꢀtheꢀphaseꢀcalculations.ꢀMapsꢀareꢀdisplayedꢀatꢀaꢀ
contourꢀlevelꢀofꢀ2ꢀσ.ꢀDistancesꢀareꢀmeasuredꢀfromꢀtheꢀhemeꢀironꢀtoꢀtheꢀcenterꢀofꢀtheꢀphenolꢀringꢀandꢀ
betweenꢀtheꢀTyr191ꢀhydroxylꢀandꢀtheꢀresidueꢀ232ꢀsideꢀchainꢀatomꢀofꢀclosestꢀapproach.ꢀDistancesꢀareꢀ
averageꢀmeasurementsꢀfromꢀtheꢀtwoꢀcomplexesꢀcontainedꢀinꢀtheꢀASU.ꢀ(D)ꢀSuperpositionꢀofꢀCcPꢀY191ꢀ
variants:ꢀW191Yꢀ(red),ꢀW191Y:L232Eꢀ(purple),ꢀW191Y:L232Hꢀ(green),ꢀallꢀinꢀcomplexꢀwithꢀCc.ꢀSubstitutionꢀ
ofꢀLeu232ꢀwithꢀGluꢀresultsꢀinꢀaꢀ1.04ꢀÅꢀdisplacementꢀofꢀTyr191.ꢀReplacementꢀwithꢀHis232ꢀdoesꢀnotꢀ
significantlyꢀshiftꢀtheꢀpositionꢀofꢀTyr191;ꢀhowever,ꢀunderꢀtheseꢀcrystallizationꢀconditions,ꢀHis232ꢀisꢀ
angledꢀawayꢀfromꢀTyr191,ꢀwhichꢀisꢀlikelyꢀaꢀresultꢀofꢀhydrogenꢀbondingꢀtoꢀThr234ꢀorꢀanꢀelectrostaticꢀ
interactionꢀwithꢀAsp235.ꢀ
263ꢀ
264ꢀ
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72ꢀ InꢀW191Y:L232E,ꢀtheꢀphenolꢀringꢀofꢀTyr191ꢀshiftsꢀcloserꢀtoꢀtheꢀ232ꢀresidueꢀbyꢀanꢀRMSDꢀofꢀ0.7ꢀÅꢀ
73ꢀ comparedꢀtoꢀW191Y.ꢀTheꢀcarboxylꢀmoietyꢀofꢀGlu232ꢀandꢀtheꢀphenolꢀofꢀTyr191ꢀareꢀwellꢀdefinedꢀ
o c
74ꢀ atꢀtheꢀ1ꢀσꢀlevelꢀinꢀtheꢀ2.9ꢀÅꢀresolutionꢀ2F –F ꢀelectronꢀdensityꢀmap.ꢀAꢀshortꢀ2.6-Åꢀdistanceꢀofꢀ
75ꢀ separationꢀbetweenꢀtheꢀTyr191ꢀhydroxylꢀgroupꢀandꢀGlu232ꢀcarboxylꢀoxygenꢀimpliesꢀaꢀstrongꢀ
276ꢀ hydrogenꢀbondꢀbetweenꢀtheꢀsideꢀchains.ꢀDifferenceꢀelectronꢀdensityꢀbridgesꢀtheꢀtwoꢀsideꢀ
277ꢀ chainsꢀinꢀbothꢀofꢀtheꢀCcP:Ccꢀcomplexesꢀinꢀtheꢀasymmetricꢀunitꢀ(Figureꢀ4B).ꢀInꢀW191Y:L323E,ꢀ
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78ꢀ comparedꢀtoꢀW191Y,ꢀtheꢀCcPꢀhemeꢀtoꢀTyr191ꢀphenylꢀringꢀdistanceꢀincreasesꢀbyꢀ0.5ꢀÅꢀ(∼7.1ꢀÅꢀinꢀ
79ꢀ W191Y:L232Eꢀversusꢀ∼6.6ꢀÅꢀinꢀW191Y)ꢀandꢀtheꢀdistanceꢀbetweenꢀtheꢀCcꢀhemeꢀironꢀatomꢀinꢀCcꢀ
80ꢀ andꢀTyr191ꢀalsoꢀslightlyꢀincreasesꢀbyꢀ0.5ꢀÅꢀ(∼21.6ꢀÅꢀinꢀW191Y:L232Eꢀversusꢀ∼21.0ꢀÅꢀinꢀW191Y).ꢀ
81ꢀ Thus,ꢀtheꢀY191ꢀandꢀE232ꢀsubstitutionsꢀdoꢀnotꢀsubstantiallyꢀalterꢀtheꢀproteinꢀcomplex,ꢀbutꢀtheꢀ
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282ꢀ structuresꢀdoꢀsupportꢀaꢀhydrogenꢀbondꢀbetweenꢀtheseꢀtwoꢀresiduesꢀpriorꢀtoꢀreaction.ꢀ
283ꢀ
ꢀ
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84ꢀ ReplacementꢀofꢀLeu232ꢀwithꢀHisꢀalsoꢀresultsꢀinꢀaꢀstructureꢀveryꢀsimilarꢀtoꢀthatꢀofꢀtheꢀW191Yꢀ
85ꢀ parent.ꢀHowever,ꢀinꢀthisꢀstructureꢀ(crystallizedꢀatꢀpHꢀ6ꢀwhereꢀETꢀratesꢀdoꢀnotꢀincreaseꢀwithꢀtheꢀ
86ꢀ His232ꢀaddition)ꢀHis232ꢀanglesꢀawayꢀfromꢀY191,ꢀandꢀsharesꢀnoꢀbridgingꢀelectronꢀdensityꢀwithꢀ
87ꢀ theꢀphenolicꢀoxygen.ꢀInstead,ꢀdifferenceꢀdensityꢀbetweenꢀTyr191ꢀandꢀHis175ꢀsuggestsꢀaꢀ
288ꢀ competitionꢀbetweenꢀHis175ꢀandꢀHis232ꢀforꢀtheꢀphenolicꢀoxygenꢀofꢀTyr191ꢀ–ꢀbothꢀnitrogenꢀ
289ꢀ atomsꢀareꢀwithinꢀhydrogen-bondingꢀdistanceꢀofꢀtheꢀTyr191-OHꢀ(His175ꢀN-δꢀ∼ꢀ3.9ꢀÅꢀandꢀHis232ꢀ
2
90ꢀ N-εꢀ∼ꢀ3.1ꢀÅ).ꢀFurthermore,ꢀtheꢀHis232ꢀprotonatedꢀNδꢀnitrogenꢀisꢀinꢀrangeꢀtoꢀinteractꢀwithꢀ
2
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91ꢀ Thr234ꢀorꢀAsp235ꢀ(Figureꢀ4D).ꢀThus,ꢀinꢀtheꢀimidazoliumꢀform,ꢀHis232ꢀcannotꢀacceptꢀaꢀprotonꢀ
92ꢀ fromꢀY191ꢀandꢀmayꢀnotꢀbeꢀsuitablyꢀpositionedꢀtoꢀhydrogenꢀbondꢀbackꢀtoꢀtheꢀTyrꢀradical,ꢀ
93ꢀ consistentꢀwithꢀanꢀETꢀrateꢀsimilarꢀtoꢀthatꢀofꢀtheꢀparentꢀatꢀlowꢀpH.ꢀHowever,ꢀatꢀhigherꢀpH,ꢀ
294ꢀ His232ꢀisꢀpositionedꢀappropriatelyꢀforꢀtheꢀneutralꢀimidazoleꢀmoietyꢀtoꢀinteractꢀproductivelyꢀ
295ꢀ withꢀTyr191ꢀtoꢀenhanceꢀreactivity.ꢀ
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96ꢀ
ꢀ
97ꢀ Theꢀresolutionꢀofꢀtheꢀstructuresꢀisꢀnotꢀsufficientꢀtoꢀdefineꢀanyꢀclearlyꢀorderedꢀwaterꢀmoleculesꢀ
98ꢀ inꢀtheꢀproximalꢀhemeꢀpocket.ꢀHowever,ꢀwaterꢀmoleculesꢀareꢀwellꢀresolvedꢀinꢀstructuresꢀofꢀtheꢀ
99ꢀ WTꢀCcP:Ccꢀcomplexꢀ(1U74)ꢀandꢀW191FꢀCcP:Ccꢀ(5CIF),ꢀaꢀstructuralꢀanalogueꢀtoꢀW191YꢀCcP:Cc.ꢀ
300ꢀ Superpositionsꢀofꢀtheseꢀstructuresꢀpredictꢀnoꢀorderedꢀwaterꢀmoleculesꢀnearꢀtheꢀionizableꢀ
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01ꢀ moietiesꢀofꢀtheꢀ191ꢀandꢀ232ꢀresidues,ꢀalthoughꢀtheꢀintroductionꢀofꢀpolarꢀgroupsꢀatꢀtheseꢀ
02ꢀ positionsꢀcouldꢀalterꢀsolvation.ꢀ
03ꢀ
ꢀ
04ꢀ ElectronꢀspinꢀresonanceꢀspectroscopyꢀofꢀTyr191ꢀradicalsꢀindicatesꢀchangesꢀinꢀhydrogenꢀbondꢀ
05ꢀ environment.ꢀ
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06ꢀ CW-ESR:ꢀX-bandꢀcw-ESRꢀwasꢀusedꢀtoꢀfurtherꢀprobeꢀtheꢀelectronicꢀandꢀprotonationꢀpropertiesꢀofꢀ
•
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07ꢀ theꢀW191Y ꢀvariants.ꢀAsꢀpreviouslyꢀreported ,ꢀtheꢀcw-ESRꢀspectrumꢀofꢀW191YꢀCpdꢀIꢀindicatesꢀ
•
308ꢀ formationꢀofꢀTyr ꢀatꢀsiteꢀ191;ꢀW191Gꢀproducesꢀaꢀnegligibleꢀsignalꢀatꢀgꢀ=ꢀ2.0.ꢀIncorporationꢀofꢀ
309ꢀ 2,3,5-FTyrꢀexhibitsꢀaꢀsignalꢀreminiscentꢀofꢀtheꢀparentꢀ(orange,ꢀFigureꢀ5A).ꢀThisꢀisꢀinꢀcontrastꢀtoꢀ
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10ꢀ theꢀlineꢀbroadeningꢀobservedꢀforꢀtheꢀphotogeneratedꢀ2,3,5-FTyrꢀradicalꢀinꢀaqueousꢀsolution
,ꢀ
11ꢀ whichꢀlikelyꢀderivesꢀfromꢀanꢀincreaseꢀinꢀanisotropyꢀandꢀhyperfineꢀinteractionsꢀfromꢀtheꢀfluorineꢀ
12ꢀ moietiesꢀinꢀaꢀfullyꢀsolvatedꢀaqueousꢀenvironment.ꢀTheꢀfluoroꢀgroupsꢀareꢀknownꢀtoꢀmoderatelyꢀ
13ꢀ reduceꢀspinꢀpopulationꢀatꢀtheꢀoxygenꢀbutꢀoverallꢀdoꢀnotꢀcauseꢀmajorꢀredistributionꢀofꢀtheꢀspinꢀ
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314ꢀ density,ꢀwhichꢀremainsꢀsymmetricꢀaboutꢀtheꢀC2ꢀrotationalꢀaxis .ꢀSubstantialꢀholeꢀmigrationꢀ
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15ꢀ fromꢀFTyr191ꢀtoꢀanꢀadjacentꢀtyrosineꢀinꢀCcPꢀisꢀunlikelyꢀasꢀradicalꢀmigrationꢀawayꢀfromꢀtheꢀ191ꢀ
16ꢀ siteꢀwouldꢀreduce,ꢀnotꢀincreaseꢀtheꢀCcꢀoxidationꢀrates.ꢀ
3
17ꢀ
ꢀ
318ꢀ
Figureꢀ5ꢀ–ꢀcw-ESRꢀofꢀCpdꢀIꢀprovidesꢀevidenceꢀforꢀhydrogenꢀbondꢀformationꢀinꢀL232E,HꢀCcP.ꢀ(A)ꢀX-bandꢀ
cw-ESRꢀspectraꢀofꢀCpdꢀIꢀacquiredꢀatꢀ12ꢀKꢀforꢀCcPꢀvariants.ꢀSamplesꢀcontainedꢀ100ꢀmMꢀKPi,ꢀpHꢀ6ꢀbuffer,ꢀ
exceptꢀforꢀwhereꢀindicated.ꢀW191Y:L232HꢀatꢀpHꢀ7.5ꢀwasꢀacquiredꢀwithꢀ4ꢀGꢀmodulation,ꢀandꢀallꢀothersꢀ
wereꢀcollectedꢀwithꢀ1.5ꢀ–ꢀ2ꢀGꢀmodulation.ꢀSpectraꢀwereꢀbase-lined,ꢀnormalized,ꢀandꢀshiftedꢀvertically.ꢀ
(B)ꢀInꢀtheꢀX-bandꢀcw-ESRꢀspectraꢀofꢀW191Y:L232E,ꢀhighꢀpHꢀbufferꢀresultsꢀinꢀnarrowingꢀandꢀdecreaseꢀofꢀ
signalꢀintensityꢀ(inset),ꢀindicatingꢀlossꢀofꢀtheꢀphenolicꢀoxygenꢀhydrogenꢀbond.ꢀ(C,D)ꢀX-bandꢀcw-ESRꢀofꢀ
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19ꢀ
20ꢀ
21ꢀ
22ꢀ
23ꢀ
24ꢀ
2 2
H OꢀandꢀD O-exchangedꢀCpdꢀI.ꢀW191YꢀCpdꢀIꢀshowsꢀonlyꢀmodestꢀchangesꢀafterꢀbuffer-exchange,ꢀwhereasꢀ
325ꢀ
326ꢀ
W191Y:L232EꢀCpdꢀIꢀexhibitsꢀaꢀnarrowedꢀlineꢀshapeꢀinꢀD
ꢀ
2
OꢀthatꢀresemblesꢀthatꢀofꢀW191YꢀCpdꢀI.ꢀꢀ
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27ꢀ CpdꢀIꢀofꢀW191Y:L232HꢀatꢀpHꢀ6ꢀshowsꢀonlyꢀminorꢀdifferencesꢀinꢀhyperfineꢀcouplingꢀcomparedꢀtoꢀ
28ꢀ theꢀparentꢀW191Y;ꢀhowever,ꢀraisingꢀtheꢀpHꢀtoꢀ7.5ꢀresultsꢀinꢀdramaticꢀlineꢀbroadeningꢀofꢀtheꢀ
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329ꢀ singletꢀfeatureꢀrelativeꢀtoꢀW191Yꢀ(darkꢀgreen,ꢀFigureꢀ5A).ꢀW191Y:L232EꢀatꢀpHꢀ6ꢀshowsꢀevenꢀ
330ꢀ moreꢀpronouncedꢀlineꢀbroadeningꢀthanꢀL232HꢀatꢀpHꢀ7.5.ꢀWhenꢀtheꢀpHꢀisꢀraisedꢀtoꢀ8,ꢀspectraꢀforꢀ
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31ꢀ W191Y:L232EꢀnarrowꢀtoꢀaꢀwidthꢀsimilarꢀtoꢀthatꢀofꢀW191Y,ꢀalthoughꢀsignalꢀintensityꢀalsoꢀlessensꢀ
32ꢀ (Figureꢀ5B).ꢀTheꢀchangeꢀinꢀESRꢀlineꢀshapeꢀinꢀpHꢀconditionsꢀthatꢀalsoꢀenhanceꢀETꢀratesꢀsignifiesꢀ
•
33ꢀ anꢀalteredꢀmicroenvironmentꢀofꢀtheꢀTyr ꢀinꢀtheꢀpresenceꢀofꢀtheseꢀcoordinatingꢀbases.ꢀForꢀ
34ꢀ His232,ꢀlineꢀbroadeningꢀrequiresꢀdeprotonationꢀofꢀtheꢀimidazoliumꢀaboveꢀpHꢀ6.0,ꢀconsistentꢀ
335ꢀ withꢀtheꢀlowꢀpHꢀcrystalꢀstructure.ꢀAꢀsimilarꢀbroadeningꢀofꢀtheꢀY191:E232ꢀspectraꢀatꢀpHꢀbelowꢀ8ꢀ
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36ꢀ likelyꢀderivesꢀfromꢀtheꢀhydrogenꢀbondꢀbetweenꢀE232ꢀandꢀY191ꢀinferredꢀfromꢀtheꢀcrystalꢀ
37ꢀ structure.ꢀInꢀadditionꢀtoꢀhyperfineꢀinteractionsꢀfromꢀhydrogen-bondedꢀprotons,ꢀtheꢀrotationalꢀ
•
38ꢀ angleꢀofꢀtheꢀphenolꢀringꢀcanꢀinfluenceꢀTyr ꢀlineꢀshapes.ꢀTheꢀdegreeꢀofꢀhyperconjugationꢀofꢀaꢀC
β
ꢀ
39ꢀ –Hꢀσꢀbondꢀintoꢀtheꢀphenolꢀπꢀsystemꢀaltersꢀrelativeꢀcontributionsꢀofꢀaꢀnarrowꢀsingletꢀandꢀaꢀwideꢀ
9
2,93
40ꢀ tripletꢀsignal
.ꢀHowever,ꢀtheꢀcrystalꢀstructuresꢀofꢀW191YꢀandꢀW191Y:L232Eꢀdoꢀnotꢀdifferꢀinꢀ
341ꢀ theꢀTyr191ꢀrotamerꢀstate,ꢀandꢀthus,ꢀchangesꢀinꢀhyperfineꢀcouplingꢀlikelyꢀresultꢀfromꢀhydrogenꢀ
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42ꢀ bondꢀformationꢀwithꢀtheꢀconjugateꢀbase.ꢀIndeed,ꢀelectronicꢀstructureꢀcalculationsꢀsuggestꢀthatꢀ
43ꢀ theꢀspinꢀdistributionꢀofꢀtyrosylꢀradicals,ꢀasꢀreflectedꢀinꢀtheꢀgꢀvaluesꢀandꢀlineꢀshapesꢀofꢀESRꢀ
94–98
44ꢀ spectra,ꢀareꢀhighlyꢀsensitiveꢀtoꢀprotonationꢀandꢀhydrogenꢀbonding
.ꢀ
45ꢀ
ꢀ
46ꢀ Somewhatꢀsurprisingly,ꢀbutꢀconsistentꢀwithꢀtheꢀreactivityꢀdata,ꢀadditionꢀofꢀaꢀcoordinatingꢀsideꢀ
347ꢀ chainꢀtoꢀtheꢀ2,3,5-FTyrꢀvariantꢀW191F
3
Y:L232Eꢀdoesꢀnotꢀbroadenꢀtheꢀradicalꢀspectrumꢀ(Figureꢀ
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48ꢀ 5A).ꢀOwingꢀtoꢀtheꢀhigherꢀacidityꢀofꢀFTyrꢀcomparedꢀtoꢀTyr ,ꢀeitherꢀaꢀprotonꢀisꢀlostꢀfromꢀtheꢀ
7
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49ꢀ systemꢀ(whichꢀisꢀlessꢀprobableꢀinꢀaꢀpHꢀ6ꢀbuffer;ꢀpK
a
ꢀ(FTyr)ꢀ= 6.4 )ꢀorꢀtheꢀhydrogenꢀbondꢀsharedꢀ
•
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50ꢀ withꢀGlu232ꢀisꢀweakerꢀthanꢀinꢀW191Y:L232E.ꢀInꢀtheꢀlatterꢀcase,ꢀaꢀlongerꢀFTyr ···Hꢀdistanceꢀmayꢀ
3
51ꢀ reduceꢀtheꢀinfluenceꢀonꢀtheꢀcouplingꢀparametersꢀofꢀtheꢀradical ꢀandꢀrateꢀofꢀET.ꢀꢀ
52ꢀ
ꢀ
•
53ꢀ X-bandꢀcw-ESRꢀmeasurementsꢀofꢀD
2
O-treatedꢀY191 ꢀshowꢀsharpenedꢀhyperfineꢀfeaturesꢀandꢀ
O-substitutionꢀforꢀ
55ꢀ W191Y:L232EꢀgivesꢀmuchꢀmoreꢀprominentꢀeffectsꢀthanꢀW191Yꢀalone,ꢀclearlyꢀdemonstratingꢀtheꢀ
354ꢀ narrowingꢀofꢀlineꢀshapesꢀasꢀexpectedꢀ(Figureꢀ5C,D).ꢀHowever,ꢀD
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56ꢀ dependencyꢀofꢀlineꢀbroadeningꢀonꢀhyperfineꢀcouplingꢀfromꢀexchangeableꢀprotonsꢀinꢀtheꢀ
57ꢀ presenceꢀofꢀE232.ꢀ
358ꢀ
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Figureꢀ6ꢀ–ꢀThree-pulseꢀESEEMꢀspectraꢀcollectedꢀatꢀ33.8ꢀGHzꢀonꢀsamplesꢀofꢀW191YꢀCpdꢀI,ꢀW191Y:L232Eꢀ
1
CpdꢀI,ꢀandꢀphotogeneratedꢀtyrosylꢀsolution.ꢀ(A,B)ꢀNormalizedꢀ H-ESE-ENDORꢀspectraꢀofꢀW191Yꢀandꢀ
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63ꢀ
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W191Y:L232EꢀCpdꢀIꢀspeciesꢀandꢀphotogeneratedꢀtyrosylꢀinꢀH OꢀandꢀD Oꢀ100ꢀmMꢀKPiꢀbuffer,ꢀpHꢀ6;ꢀꢀνLarmorꢀ
∼51ꢀmHz.ꢀSubtractedꢀspectraꢀofꢀCpdꢀIꢀspeciesꢀ(inꢀdarkꢀandꢀlightꢀgrey)ꢀinꢀprotonatedꢀandꢀdeuteratedꢀ
environmentsꢀrevealꢀH-bondingꢀinteractions.ꢀSpectralꢀinterferenceꢀwithꢀotherꢀweaklyꢀboundꢀprotons,ꢀ
however,ꢀmakesꢀquantificationꢀdifficult.ꢀTheꢀasymmetricꢀshoulderꢀfeatureꢀinꢀtheꢀW191YꢀCpdꢀIꢀdifferenceꢀ
spectrumꢀisꢀlikelyꢀnotꢀsignificant.ꢀ(C)ꢀTimeꢀdomainꢀspectraꢀ(verticallyꢀstackedꢀandꢀamplitudesꢀnormalizedꢀ
fromꢀ0ꢀtoꢀ1ꢀbetweenꢀ0ꢀnsꢀandꢀ3000ꢀns)ꢀexhibitꢀweakerꢀstimulated-echoꢀamplitudeꢀforꢀW191YꢀCpdꢀIꢀ
comparedꢀtoꢀsimilarlyꢀpreparedꢀsamplesꢀofꢀW191Y:L232EꢀCpdꢀIꢀandꢀphotogeneratedꢀTyr.ꢀ(D)ꢀFourier-
369ꢀ
370ꢀ
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transformedꢀsignalsꢀshowꢀhyperfineꢀcouplingsꢀnearꢀthatꢀofꢀ HꢀZeemanꢀfrequency.ꢀSignalsꢀnormalizedꢀtoꢀ
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71ꢀ
72ꢀ
73ꢀ
theꢀechoꢀamplitudesꢀatꢀ0ꢀnsꢀdemonstrateꢀtheꢀlowerꢀlevelsꢀofꢀhydrogenꢀbondingꢀinꢀW191YꢀCpdꢀIꢀ
comparedꢀtoꢀW191Y:L232E.ꢀꢀ
ꢀ
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74ꢀ Electronꢀspinꢀecho-electronꢀnuclearꢀdoubleꢀresonanceꢀ(ESE-ENDOR)ꢀspectroscopy:ꢀꢀ
75ꢀ Q-bandꢀESE-ENDORꢀdataꢀonꢀprotonatedꢀandꢀdeuteratedꢀW191Yꢀ(Figureꢀ6A)ꢀandꢀW191Y:L232Eꢀ
•
376ꢀ (Figureꢀ6B)ꢀCpdꢀIꢀsamplesꢀsupportꢀinteractionsꢀbetweenꢀtheꢀTyr ꢀradicalꢀandꢀexchangeableꢀ
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77ꢀ protons.ꢀTheꢀspeciesꢀinꢀ HꢀbufferꢀhaveꢀsimilarꢀprotonꢀESE-ENDORꢀspectraꢀandꢀweakꢀhyperfineꢀ
78ꢀ couplingꢀfeaturesꢀcenteredꢀatꢀtheꢀLarmorꢀfrequencyꢀ(∼51ꢀMHz)ꢀthatꢀlikelyꢀoriginateꢀfromꢀ
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79ꢀ exchangeableꢀprotonsꢀ(redꢀandꢀpurpleꢀspectra;ꢀFigureꢀ6A,B).ꢀAfterꢀincubationꢀinꢀdeuteratedꢀ
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80ꢀ buffer,ꢀbothꢀspeciesꢀ(blueꢀandꢀgreenꢀspectra;ꢀFigureꢀ6A,B)ꢀexhibitꢀsimilarꢀ H-hyperfineꢀcouplingꢀ
381ꢀ constantsꢀtoꢀthoseꢀofꢀphotogeneratedꢀtyrosylꢀstandardsꢀinꢀdeuteratedꢀsolutionꢀ(greyꢀspectrum;ꢀ
382ꢀ Figureꢀ6A,B).ꢀTheseꢀnonexchangeableꢀfeaturesꢀlikelyꢀresultꢀfromꢀaꢀcombinationꢀofꢀhyperfineꢀ
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83ꢀ couplingꢀinteractionsꢀbetweenꢀtheꢀunpairedꢀradicalꢀandꢀβ-methyleneꢀorꢀringꢀprotonsꢀandꢀhaveꢀ
2
•ꢀ
84ꢀ beenꢀassignedꢀusingꢀESE-ENDORꢀandꢀ H-labelingꢀinꢀotherꢀTyr systems,ꢀsuchꢀasꢀY
D
ꢀandꢀY
.ꢀSubtractionꢀofꢀtheꢀ HꢀESE-ENDORꢀofꢀtheꢀprotonatedꢀandꢀ
86ꢀ correspondingꢀdeuterium-exchangedꢀsamplesꢀ(blackꢀspectra;ꢀFigureꢀ6A,B),ꢀscaledꢀbyꢀtheꢀ
Z
ꢀinꢀPSꢀIIꢀ
99–103
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85ꢀ andꢀTyrꢀsingleꢀcrystals
387ꢀ unchangingꢀshoulderꢀfeaturesꢀatꢀνꢀ=ꢀ±ꢀ0.3ꢀMHz,ꢀrevealsꢀcontributionsꢀfromꢀexchangeableꢀ
•
•
•
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88ꢀ protonsꢀcoupledꢀtoꢀTyr191 .ꢀTheꢀdifferenceꢀspectraꢀindicateꢀthatꢀbothꢀtheꢀY191 ꢀandꢀY191 :E232ꢀ
89ꢀ radicalsꢀhaveꢀsomeꢀdegreeꢀofꢀH-bonding,ꢀyetꢀowingꢀtoꢀtheꢀspectralꢀnoiseꢀinꢀtheꢀspectra,ꢀ
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90ꢀ broadeningꢀeffectsꢀareꢀnotꢀwellꢀdetermined.ꢀTypically,ꢀbroadenedꢀ H
91ꢀ beenꢀattributedꢀtoꢀH-bonding,ꢀasꢀobservedꢀbetweenꢀPSꢀIIꢀY ꢀandꢀY ꢀ(bothꢀH-bonded)ꢀandꢀE.ꢀcoliꢀ
92ꢀ RNRꢀY122ꢀ(notꢀH-bonded)ꢀtyrosylꢀsignals .ꢀTheꢀY191:E232ꢀdifferenceꢀspectraꢀdoesꢀappearꢀ
2 2
Oꢀ−ꢀ H Oꢀsignalsꢀhaveꢀ
Z
D
1
04
393ꢀ broaderꢀthanꢀY191ꢀalone,ꢀbutꢀsuchꢀsignalsꢀareꢀdifficultꢀtoꢀresolveꢀandꢀwouldꢀlikelyꢀbenefitꢀfromꢀ
1
3
3
3
3
3
94ꢀ higher-fieldꢀspectroscopy.ꢀꢀToꢀimproveꢀuponꢀinterpretationsꢀfromꢀnon-idealꢀ HꢀENDORꢀspectralꢀ
2
95ꢀ subtractions,ꢀweꢀalsoꢀattemptedꢀtoꢀobtainꢀdeuteriumꢀESE-ENDORꢀspectra.ꢀHigh-resolutionꢀ H-
•
96ꢀ ESE-ENDORꢀspectraꢀofꢀH-bondedꢀTyr ꢀradicalsꢀoftenꢀproduceꢀsharpꢀpeaksꢀorꢀ“wings”ꢀ<ꢀ0.6ꢀMHzꢀ
104–108
2
97ꢀ fromꢀνLarmor
.ꢀBothꢀY191ꢀandꢀY191:E232ꢀCcPꢀCpdꢀIꢀrevealꢀtheꢀpresenceꢀofꢀexchangeableꢀ H,ꢀ
98ꢀ butꢀunfortunately,ꢀpoorꢀS/Nꢀratiosꢀpreventꢀquantificationꢀandꢀlimitꢀanalysis.ꢀꢀ
399ꢀ
ꢀ
4
4
4
4
4
00ꢀ Electronꢀspinꢀechoꢀenvelopeꢀmodulationꢀ(ESEEM)ꢀspectroscopy:ꢀꢀ
2
2
01ꢀ
H-ESEEMꢀspectroscopyꢀallowsꢀforꢀtheꢀquantificationꢀofꢀdipolarꢀcontributionsꢀbetweenꢀ Hꢀandꢀ
•
02ꢀ Tyr ꢀusingꢀhyperfineꢀinteractionsꢀfromꢀtheꢀobservationꢀofꢀtheꢀstimulatedꢀechoꢀandꢀprovidesꢀanꢀ
2
03ꢀ importantꢀcomplementꢀtoꢀtheꢀESE-ENDORꢀresults.ꢀFurthermore,ꢀ H-ESEEMꢀhasꢀhighꢀsensitivityꢀ
04ꢀ towardꢀweak,ꢀanisotropicꢀcouplings,ꢀandꢀtheꢀechoꢀintensitiesꢀareꢀinfluencedꢀbyꢀtheꢀdistanceꢀ
2
405ꢀ betweenꢀelectronꢀandꢀ Hꢀspins,ꢀprovidingꢀtheꢀaddedꢀadvantageꢀofꢀspatialꢀselectivityꢀforꢀcoupledꢀ
1
09–114
2
4
4
06ꢀ nucleiꢀwithinꢀ1ꢀnmꢀofꢀtheꢀradical
.ꢀNotableꢀdifferencesꢀareꢀfoundꢀinꢀtheꢀ H-ESEEMꢀspectraꢀ
07ꢀ betweenꢀtheꢀW191Y:L232EꢀvariantꢀandꢀtheꢀW191YꢀparentꢀafterꢀexchangeꢀintoꢀD
2
Oꢀ(Figureꢀ
18
ꢀ
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