Switches “clásicos” y “cuánticos” en transferencia

electrónica biológica

Laboratorio de Biofisicoquímica

Departamento de Química Inorgánica - INQUIMAE

FCEN-UBA / CONICET

Daniel Murgida

The electrons of life

Cytochrome c

Cytochrome c oxidase (CIV)

Protein electron transfer (ET) theory

High temperature limit of semiclassical equation

Marcus Theory

RT

GH

RThk DAET

4exp

420

2

2

3

solvproti

00 EnFG

k

S

k

j

HB

j

i

C

iDAH

2exp0

dHHDA

Pathways Model

Beratan, Gray, et. al. and subsequent refinements

Some requisites of ET cascades

Goals of photosynthesis and respiration: Electro-protonic energy transduction

•Need for small ET driving forces

(downhill cascade of closely

spaced redox potentials)

•But sufficiently fast ET kinetics

for sustaining life

•Directionality

RT

GH

RThk DAET

4exp

420

2

2

3

Evolutionary design must compatibilize both requisites

A few building blocks to cover a broad E0 range

RT

GH

RThk DAET

4exp

420

2

2

3

Fine Tuning of redox potentials

M160H 148mV

M160Q 158mV

M160Y 348mV

M160S 209mV

M160L 346mV

WT 293mV

CuA center

J. Am. Chem. Soc. 2007, 129, 11884.

Tyrosine (Y) Lysine (L) Serine (S) Glutamine (Q) Histidine (H)

Electric field effects in protein ET?

Conditions are different from solution:

High electric fields (up to 109 V/m) Protein structure

Dipoles alignment

Dipole induction

Alteration of pKa´s

Activation energies

Dielectric constants

Energy levels

S S S S S S S S

Electrostatic (CO2-, PO3

2-, NH3+)

S S S S S S S S

Hydrophobic (e.g. CH3)

S S S S S S S S

Mixed SAMs (e.g. OH/CH3)

S S S S S S S S

Polar (e.g. OH)

S S S S

S S S

S

O

N H

Covalent (cross linking)

S S S S

S S S

S O

O

N

Coordinative (e.g. Py)

Acc. Chem. Res. 2004, 37, 854; PCCP 2005, 7, 3773; Chem. Soc. Rev. 2008, 37, 937; PCCP 2013, 15, 5386; Langmuir 2013, in press

His-tag/Ni-NTA

Detergent mediated

Model systems

Electric field strength

Electric field ca. 108-109 V / m

Reporter group

Vibrational Stark effect

(SER and SEIRA)

ele

ctr

ode

fM

E / V

fC

fRC

dC dRC fS

d / Å

SSSSSSSS

Fe

SAM

e- protein

CN

Ag-nanocoral Ag-nanopillars

Ag – Au hybrids

Nano Lett., 2009, 9, 298; Langmuir, 2008, 24, 1583; Langmuir, 2007, 23, 11289.

SER-Active Electrodes

2

LiL BI

2

sss BI

SERR / SEIRA Spectroelectrochemistry

SERR

Heme

SEIRA

Protein

Angew. Chem. Int. Ed. 2001, 40, 728; PCCP 2008, 10, 5276; Acc. Chem. Res. 2004, 37, 854; Chem. Soc. Rev. 2008, 37, 937; JACS 2008, 130, 9844

PFV

J. Am. Chem. Soc. 2007, 129, 11884

Chem. Phys. Chem., 2010, 11, 1225

PCCP, 2008, 10, 5276

J. Phys. Chem. B 2006, 110, 19906

TR-SERR

Heme

TR-SEIRA

Protein

Nano Lett., 2009, 9, 298; Angew. Chem. Int. Ed. 2001, 40, 728; PCCP 2008, 10, 5276 Acc. Chem. Res. 2004, 37, 854; Chem. Soc. Rev. 2008, 37, 937

Protein Film Voltammetry

JACS 2007, 129, 11884; ChemPhysChem 2010, 11, 1225

PCCP 2008, 10, 5276; J. Phys. Chem. B 2006, 110, 19906

SERR / SEIRA Spectroelectrochemistry

Distance-dependence of the ET rate

dxx

N

RTx

RT

FN

N

RT

h

Hk

A

A

A

ABET

)exp(1

4exp

4

4

2

22

Long chains:

Normal distance dependence

) exp( ) ( d A d k ET

~ 1.1 / CH2

Short chains:

Distance independent

Another rate limiting step

(gating?)

S S S S S S S S

Acc. Chem. Res. 2004, 37, 854; Chem. Soc. Rev. 2008, 37, 937

J. Electroanal. Chem. 2011, 660, 367

dxx

N

RTx

RT

FN

N

RT

h

Hk

A

A

A

ABET

)exp(1

4exp

4

4

2

22

Distance-dependence of the ET rate

Electric-Field Controlled Protein Dynamics, i.e. Electronic Coupling?

Acc. Chem. Res. 2004, 37, 854; Chem. Soc. Rev. 2008, 37, 937; J. Electroanal. Chem. 2011, 660, 367

Molecular Dynamics Simulations

Electrochim Acta 2009, 54, 4963; ChemPhysChem 2010, 11, 1225; JACS 2010, 132, 5769.

70 80 90 100 110 120 130 140

140

150

160

170

180

190

200

f

Cyt+3

-100

-80

-60

-40

-20

70 80 90 100 110 120 130 140

140

150

160

170

180

190

200

f

Cyt+2

2e-4

4e-4

6e-4

8e-4

Binding Coupling

Direct observation of the gating step

400 450 500 550 600

0.0

0.5

1.0

1.5

Ab

so

rba

nce

Wavelength (nm)

413 nm

514 nm

Ag

Fe

A1g, B1g

(4, 10)

Ag

A1g

(4) Fe

Fe2+

Fe3+

Weak Field Strong Field

70 80 90 100 110 120 130 140

140

150

160

170

180

190

200

f

Cyt+3

-100

-80

-60

-40

-20

70 80 90 100 110 120 130 140

140

150

160

170

180

190

200

f

Cyt+2

2e-4

4e-4

6e-4

8e-4

Binding Coupling

S S S S S S S S

JACS 2008, 130, 9844; JACS 2009, 131, 16248; JACS 2010, 132, 5769; J. Phys. Chem. B 2013, in press

Electrostatic modulation of

Electric Field (eV)

High (strong complex)

0.37

Medium (weak complex)

0.51

Low (in solution)

0.60

Cyt-c

JACS 2013, 135, 4389

Weak Cyt/SAM electrostatic complex Strong Cyt/SAM electrostatic complex

SAM

RR solution

SERR adsorbed

WT

WTrec

Electrostatic tuning of trough 2nd sphere ligand?

Zhou et. al. Proteins Struct. Funct. Bioinf. 2009, 76, 151

H-bond scoring distribution

SAM10%

SAM25%

SAM50%

JACS 2013, 135, 4389

Electrostatic tuning of trough 2nd sphere ligand?

WT

WTrec

Y67F

Y67F mutation

Tyrosine (Y) Phenylalanine (F)

WT

WTrec

Y67F

UV-vis absorption Q-band SERR

Soret-band SERR

Spectroelectrochemistry Electrochemistry

WT

WTrec

Y67F

JACS 2013, 135, 4389

Electrostatic tuning of trough 2nd sphere ligand?

Protein film voltammetry TR-SERR

WT

Y67F

Y67F WT

Y67F

C15COOH:C15CH2OH

Y67F / C15COOH:C15CH2OH 0.41 eV

Y67F / C15COOH 0.41 eV

WT / C15COOH:C15CH2OH 0.51 eV

WT / C15COOH 0.37 eV

JACS 2013, 135, 4389

Electrostatic tuning of trough 2nd sphere ligand?

MD simulations

1 2 3

JACS 2013, 135, 4389

Specific electrostatic

(and hydrophobic)

interactions

Summary

RT

GH

RThk DAET

4exp

420

2

2

3

Unspecific

Electric Fields

Cyt

Alternative “native” structures

Altered Dynamics

)( 00 EG DAH

Modulation of

Similar effects for the electron acceptor CuA?

CuA

CuB

Heme b

Heme a3

Thermus thermophilus ba3 enzyme SII: CuA soluble fragment

The ba3 O2-reductase from Thermus thermophilus

M160

Q151

C149

C153

H114

H157

Cytochrome c

Cytochrome c oxidase (CIV)

CuA

Heme a Heme a3

CuB

Mammalian O2-reductases

CuA site

Bacterial O2-reductases

The CuA redox site

Fine tuning of electronics, dynamics and thermodynamics: weak axial ligand Met 160

Tyrosine (Y) Lysine (L) Serine (S)

Glutamine (Q) Histidine (H)

Resonance Raman

d (Å)

Cu-N Cu-S Cu-Cu

WT 1.978 2.303 2.472

M160H 1.965 2.269 2.446

M160Q 1.946 2.286 2.429

Cu K-EXAFS

JACS 2007, 129, 11884; PNAS 2012, 109, 17348; Chem. Comm. 2013, in press.

1H, 13C, 15N Paramagnetic NMR

Single Point Mutants

● Slight perturbation of His114 and His157 spin density

● Slight perturbation of Cys149 and Cys153 equivalence

● But Structure and MV character largely preserved in M160 mutants

WT 293mV

M160H 148mV M160Q 158mV

M160Y 348mV

M160S 209mV

M160L 346mV

PFV

EPR

010.2g

200.2// g

Ground state wave function

T-dependence of NMR d: β-1H of Cys153 ; α-13C of Cys149

Protein E(GS u)-E(GS su*) u population

M160Q 900 cm-1 (10.8 kJ mol-1) 1 %

WT 600 cm-1 (7.2 kJ mol-1) 5 %

M160H 200 cm-1 (2.4 kJ mol-1) 30 %

PNAS 2012, 109, 17348; Chem. Comm. 2013, in press.

Glutamine (Q) Histidine (H)

Ground state wave function

HOMO su*

HOMO u

PNAS 2012, 109, 17348; Chem. Comm. 2013, in press.

Direct determination of for su* and u states

RAMO exp (eV) calc (eV)

su* 0.4 0.3

u 0.6 0.6

Redox

active

Redox

inactive

RT

GH

RThk DAET

4exp

420

2

2

3

PNAS 2012, 109, 17348; Chem. Comm. 2013, in press.

ET pathways

Optimal pathways Coupling decay

Inter-protein ET: Cyt552 to CuA

HEC-MET69-VAL68-MET160-CuA 7.4x10-5

Intra-protein ET: CuA to heme b

CuA-HIS157-ARG450-HEM 2.3x10-5

Atom Contribution to HOMO wavefunction

Cu1.5-Cu1.5 Cu1-Cu1

su* u su* u

NHis157 0.00 0.02 0.02 0.01

SMet160 0.01 0.11 0.02 0.01

Electron

entry

Electron

exit

PNAS 2012, 109, 17348; Chem. Comm. 2013, in press.

RT

GH

RThk DAET

4exp

420

2

2

3

Modulation of the GS wavefunction

Cu1.5-Cu1.5

Cu1-Cu1

su* u

Cu1.5-Cu1.5

su* u

Cu1.5-Cu1.5

su* u

u

su*

Cu1.5-Cu1.5

No Field EF along S-S EF along Cu-Cu EF ┴ Cu2S2 core

PNAS 2012, 109, 17348; Chem. Comm. 2013, in press.

Summary / Hypothesis

EF Regulation of the Respiratory Function

High EF

DQIAyQF - INQUIMAE

Ulises

Zitare

Daiana

Capdevila Waldemar

Marmisollé

María A.

Castro

María F.

Molinas

Damián

Álvarez-Paggi

Daniel

Murgida

Agradecimientos

Colaboraciones

IBR-Argentina

Alejandro Vila

Gabriela Ledesma

Luciano Abriata

UNR-Uruguay

Rafael Radi

Veronica Tórtora

Verónica Demicheli

Financiación

AvH-Stiftung

CONICET, ANPCyT, UBA,

CeBEM