Redox kinetics of quinones in lipid monolayers

:-.

_.

The redov equilibria and kinetics of- the quindnes : I-V&m& ,_Kr- .. (VK,) undecyl-dihydroxy-benzo@rinone LUB_Q(OH) J and di-&_ti+i~j+: benzoquinone (DBBQ) incorporated intb lipid mcnolayers :and bilayer.. : vesicles ivere investigated by cyclic voltammetry. .. Deviation- -from_equ&~ _ librium of the redtiction and the oxidation -potenti_als.oS-.the quip&& in-_ the lipid monolayers was induced by condensingthe rnonolay~~._.~.Addit~~~.-‘of the uncoupler carbonyl-cyanid~z-chlorophenyl-hydiazcne: (C_C_CP) rep_i verted the condensation effect and shifted the sy&em --back- tdaPards~::l equilibrium. -The redox potential of VK, and DBBQ shifted;’ as&pect$d,?‘G by 55-60 mV per pH unit: UBQ(OH), 3boked a +&y l&ge:&ift k.it$r&d~x~-I ‘Such_ a &ift indicat~s;~h~~“~~~~_: potential of about IOO mV per pH unit. uptake of protons by the cluinone during its -oxid’ationis largerth& &e proton per electron_ The measured low pK’s of icniiatio’i; bf TII@{~~j~, _ as compared with pK’s of UBQH,(OH), is consistent.. with_: rbe -hrgh=p_rE;I. ton uptake during reduction. *. .

Introduction

.-_ ._

_.

-_ .x

..-.

-~

._

. _*-_

_-

_-

.

:,

One way to get. an idea about the position of- different: coml5onentS r in the electron transfer chain in mitochondria,‘ ~chlor&pl&t~-r&k&.‘:the’other membrane systems is to determine their redox -potentials, I :.T& -. may. be done in the intact systems, in subunits obtained after .their$$.‘: integration or in different- aqueous ektracts containing s&S-’ and$(or)‘.~ In most cases mediators are used to -'detcrmiiret$_e;red~2~: detergents. .-.- _ potentials in a particular system. It has been pointed out -by. many invcstigatorsi*2 .thSt th&&do$~;~ potential depends very significantly on the ~_microenvironment=~of%i
Miller and Silverstein

264

low concentrations with regard to the redos system and different mediators have to be tested to render equal potentials. If only electrons without protons are transferred in a redox reaction,- the mrd-potential * r/;,l increases by the difference in potential between the redox system and the solution_ This is not the case when an electron and a proton are added simultaneously to the osidized form. and the electrostatic contribution is thus cancelled. The pH dependence of the redox potential and of U,,, is usually an indication that a proton is transferred together with an electron in the redos reaction_ However, U,,, may be pH dependent also without the binding of proton by the reduced form if the particle containing the redos system contains also groups which can be protonated or dissociated, thereby changing the potential of the particle_ The potentiometric titration of an ionizable group at a potential Q is given by

(1) Since, however, then dU,,, dpH = -

RT

I _

F

d kM=41

-

dPH

(3

>

where a indicates the degree of dissociation of the carbosylic group. If 4) does not vary with z, d log [oL/(I-z) ] dpH eq ua 1s unity and dU,/dpH = o. If, as in a polyacid, only the ionization of the carbosylic groups is responsible for the electrostatic potential, d log [oL/(I-a)] dpH may, between a = 0.05 and 0.95. assume values close to 0.5 depending on alt concentration,3 and dU,,, ___-dpH

RT zjli’

(4)

WILSOK and DUTTOX’ pointed out that during the electron transfer process, equilibrium does not have to be preserved and the oxidation and reduction potentials may deviate from the equilibrium redos potential_ This is also true with respect to the pH dependence of U,,E_ The solubility of a proton in the hydrophobic part of the membrane is by many orders of magnitude lower than that of water. The only source of

* This

quantity

is usuaily

employed

for a potential at equal concentration is usuallv measured at a steidy state, and

may

or may

not

coincide

with

in electron

transfers

of the oxidized and reduced but it may also be measured

the

equilibrium

potential.

in membranes form G,,,. It in transients

Redos Kinetics of Quinones in Lipid Monolayers

265

protons required by groups bein g reduced in this hydrophobic domain is by dissociation of the water molecules. Equilibration with the external acqueous solution at a given pH present in the system will be a slow subsequent process. As a result, the reduction potential in this relatively fast reduction process will. be independent of the pH of the solution_ To. transfer an electron from a donor to- an acceptor molecule, the two molecules have to be properly located and oriented with respect to each other. If only a small fraction of the electron acceptor molecules are properly oriented with respect to the donor and the rate constant for the transition from the random orientation to the actk~e orientation is kor, then the reduction rate will be given in analogy to electron transfer on an electrod” by deR = dt

-

k,, ko,,d

(5)

(I-OR) esp

where de,/ dt is the reduction current (Ired) minus the osidation current (I,,,) in number of charges per accepting site per s and ORis the fraction of the electron acceptor~in the reduced form. Here the role of the electrode is taken over bv an esternal abundant redos system whose redos potential and compo&ion determine the value of U_ korcdand ko,, are the reduction and the oxidation rate constant, a is the transfer coefficient having values ranging from zero to unit (o + 1).5~6 If k,, is very small it can convert a close to equilibrium process to a completely irreversible one. In the case of equilibrium, where the net change in degree of reduction (de&%) is very small with respect to the o_xidation current or the reduction current, IO,

In this case

Ired =

10

(6)

-_ 8 r_e

and for

=

FU

esp RT

- ko;,n = ___ = kox

h-

(64 .

8 = 0.5 UO, = (RT/P) In k’. For finite values of dOR/dt (z.e.. denjdt # o)

deR dt=

i O

a(U” - U)F RT I

(r-4

w”

-

UP

(7)

where UO is the equilibrium potential_ and U is the potential for identical values of OR and some finite value of (deR/dt). In a S~a~iOIXUy state in a membrane we have to assume the occurrence of a cyclic process which can be depicted in its simplest form as follows :

Miller and Silverstein

266

On one side of the membrane we have a reducing agent and on the other side an osidating one ; both are abundant enough to determine the osidation and the reduction potentials of the membrane redos system on the two sides of the membrane_ Equation (7) can be utilized for the osidation or the reduction reaction_ Let us consider two limiting cases for this equation :

Small values of (UO-V), and hence also of (d0Rldt). current (2) RelativelJF high xralues of (deR/dt), but with the osidation neglected. In the first case the reduction current is linearly dependent on the difference between the actual and the reversible potential (I)

deR

1

--z--=O

g%tz--“*) l_---RT

(W

For 8 = 0.5 we obtain with the aid of equation (6)

= u”,, Thus

the combined

0.5 Fk,, k,

rate constants

_

(1-X) F u”m

RT (dkz/dt) exp

can be obtained

RT

I

in this region

from

the dependence of Unron dOR/dt. In the second case, for 6 = 0.5, equation (7) yields

There is a similarity between the redos processes in a lipid membrane and those in a lipid monolayer on an electrode surface. We have found that the redos systems embedded in a lipid monolayer are convenient for the investigation of the‘ relation between the -reduction, or oxidation currents, and their potential.

Redos

Kinetics

of

Quinones

in Lipid

Monolayers

267

A lipid monolayer at the mercuryjwater interface around the zero charge point -(electrocapillary maximum) is oriented, similarly to the air fwater interface, with the polar groups toward the aqueous phase. At potentials of around 500 mV, positive or negative with respect to the zero charge potential (zero field across the monolayer), reorientation and eventually breakdown of the monolayer structure occurs7+ Vitamin K, and undecyldihydroxy-benzoquinone, comprising quinone groups attached to hydrocarbon chains, will orient themselves when incorporated into lipid monolayers, preferably with the quinone residues towards the aqueous solution_ However, they can be reduced only when in contact with the mercury electrode surface. Moreover, in the pH region where the product of the reduction is the protonated hydroquinone the presence of a proton at the electrode surface is essential. In 2,5-dibutylbenzoquinone the quinone group is embedded within the hydrocarbon residues, which should facilitate its movement within the lipid layer_ The results of the investigation of reduction kinetics of vita&n I<,, ‘of undecyl-dihydrosy-benzoquinone and of 2,5-di-tert-butyl-benzoqmnone incorporated in lipid monolayers and in lipid bilayer vesicles, will be related in this study to their orrentation and mobility within the lipid layer.

Experimental

The phospholipids, phosphatidyl serine monosodium salt from bovine spinal cord (PS) and phosphatidyl choline-egg lecithin (PC) were Oleyl alcohol purchased from LIPID PRODUCTS, Nutfield, England. (0-A) was obtained from APPLIED SCIEXCE LABORATORIES Inc., State College, Pa. USA. Vitamin K, (VI(,) component of a-methyl-3-phytyl-r,+naphthoquinone (the phytol residue is 3,7,rI-r5-tetramethyl-2-hesadecane) was a product of MERCK, Darmstadt, TV. -Germany. 2.5-Di-tert-butyl-r,4-benzoquinone (DtBQ) and z,z-dihvdroxy-3undecyl-r,+benzoquinone [UBQ(OH).J were purchased from P”!ALTZ and BAUER. Stamford, Corm. USA_ Carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP) was a gift of Du PONT DE NEMOURS Inc., Wilmington. Delaware. USA_ All these reagents were of high purity and mere used without further purification. Water was twice distilled. The solvents hesane and benzene (spectroscopy grade) were redistilled before use. The salts used were ANALAR grade. Pre@tmtions

of the qlcinone-contnin~.~t~

lipid

systems

The lipid monolayers containg quinone were spread from benzene In the case of phospholipids, the solutions were or hesane solutions.

Miller

265

and

Silverstein

prepared after removal of the chloroform methanol solvent by a stream of nitrogen_ For the preparation of bilayer vesicles the hexane solutions were evaporated in a stream of nitrogen. After addition of water the mixture was agitated for several minutes on a vortex blender and then sonicated for 15 minutes in an N, atmosphere with a B-12 BRANSON- sonifier.

Cyclic voltammetry was employed to investigate the electrode kinetics of the different quinones embedded within the lipid layers. The measurements were performed with a CHEMTRIX Model SsP-I polarographic analyser, which comprises the TECTROSI-x 564 storage oscilloscope equipped with a CHEJITRIS-300 polarographic amplifier and polarographic time base plug-in units. The reduction and osidation processes were measured on a D.31.E. in contact with the monolaver from the gaseous phase. The method was described in a previous publication.’ where it was also shown that equilibrium prevails between the surface phases at the airlwater and the mercury[water interfaces_ An AgI_.QCl electrode in I M KC1 salt bridge was used as a reference, and a platinum gauze as an auxiliary electrode. The polarographic currents between the mercury and the platinum electrodes were measured. For better accuracy the potential between the mercury and the reference electrode was recorded with a KEETHLEY electrometer. Results Q&nones

in moxolayevs

In dilute monolavers the molecules have a high degree of freedom to rotate and the electrode surface is freely accessible to the electroactive quinone groups. As a consequence, the potentials of the cathodic reduction peaks do not differ from the anodic oxidation peaks. As one can seem from Fig. I, the reduction and osidation potentials are the same in a mixed monolayer of VI<, with oleyl alcohol and with phosphatidylserine (PS). As a matter of fact, the peak potentials are also the same in the pure VI<, monolayer and in the monolayer mixed with PC. The potentials do not depend on the ratio of VK, to the other lipids in the monolayer. The change in the scan rate from I V/s to 5 V/s shifts the peak potentials by less than IO mV_ In Fig_ z the reduction and the osidation peak potentials of VI<, in dilute monolayers of different composition are given as a function of pH. All points obtained for different monolayer compositions are on a straight line with a slope of 56 & 2 mV/pH. It is therefore evident that within the whole region between pH 5 and 12, the redox potential of VI& is independent from the monolayer composition_ The pH dependence also indicates that during the reduction nearly every electron uptake is accompanied by a proton uptake.

Redos

Kinetics

of

Quinones

in

5

-600

Lipid

Nonolayers

7

I

269

9

1

11 P”

a

-

Fig. 2. Fig. 1. Fig_ r. Voltammograms of VK, in mixed monolayers (I : I m/w) a. with oleyl alcohol (OX) serine (PS) dashed line 0.06 pg/cm’. solid line 0.21 &cm* ; b. with phosphatidyl dashed lines fqf/cmz. solid lines 0-16 pg/cm=. supporting electrolyte 0.x X NaNO, at PH 7Fig. a. Dependence of anodic (oxidation) peak potentials (full symbois) and of cathodic ones (open symbols) of Vi<. on pH 0 0 pure VI< monolayer, 5 A I : I mixed monolayer with OX. 7 v I : I monolayer with PS 0 m I : I monolayer with PC.

At increased surface concentrations, the voltammetric current peaks show some irreversible features. The irreversibility is in every case more pronounced on the cathodic_ branch of the voltammogram where the peak potential-can be shifted by hundreds of millivolts even at low sweep In the condensed region there is a dependence pn the rates (Fig. I). nature of the inert lipid in the mixed monolayer. The sizes and thelpotentials of the split pseudocapacitance peaks are not fully reproducible, and they probably reflect the non-homogeneity of the monolayer above the collapse pressure. In very case the respective peaks shift towards more negative potentials as the sweep rate increases_ The anodic (oxidation) pseudocapacitances are only slightly shifted away from their equilibrium value. The difference between the reduction and the oxidation pseudo-capacitance peaks may result from the. proton requirement of the reduction process. If the proton requirement assumption is correct, an uncoupler should turn the irreversible peak potentials back toward reversible values. For this purpose carbonyl-cyanide in chlorophenylhydrazone (CCCP) at concentrations of the order of IO-~ _M was used. It was most effective on VK1 in -mixed monolayers with oleyl alcohol. It was considerably less effective when VK, was incorporated into PC

Miller

3-p

and

Siiverstein

monolayers. and almost ineffective in PS monolayers. On the whole, the effect of CCCP was only qualitatively reproducible, and the extent of the effect varied from experiment to esperiment. Some typical results are presented in Fig. 3_ The effect of CCCP on the peak potentials of the other quinones in condensed monolayers was less pronounced. However, also the other quinones deviated less from reversibility to begin with. Undecyl-dihydrosyl-benzoquinone in dilute lipid monolayers gives and reduction peaks independent from also reversible osidation monolayer composition. A small irreversible reduction peak is also observed (Fig 4)_ Its area is only about 5 vi; of the area of the main peak, and it could be an impurity. The dependence of the peak potentials on pH is much steeper about IOO mV per pH unit. This dependence, which indicates an uptake of almost z protons per electron, has no straightforward esplanationAn explanation based on lower pk’ values of the phenolic groups in the quinone than in the hydroquinone will be given in the discussion_ In condensed monolayers the reduction and the osidation pseudocapacitance peaks are split into two nearly reversible peaks (Fig. 4)- It could be presumed that the more negative peak corresponds to the redox potential of a semiquinone.

-0.8

-0.4

3

_i

40

80

120

l-

Fig. _+_ Fig 3_ Fig. 3. Cathodic ([7 I) and anodic (0 e) peak potentials of IO y0 Vk’, in a monolayer of OA_ after each step of increase in surface concentration and of increase in concentration cf added uncoupler CCCP to the bulk phase. Scan rates I I V/s (full lines open symbols) 5 V/s (dashed lines full symbols). Fig. 4_ in I I I monolayer Voltammo,oTams of UBQ(OH), solid line o-15 vg[crn’ scan rate 2 \‘/s pH = 7_

with 0-X dashed lines 0.09 pg/crn=

The -pH dependence. of the quinones incorporated in’ bilayer ‘&i&& i $f I hi& &i&~~~&~_‘ is similar to that- _of dilute monolayers.. ~oyrey& centrations and after long -exposures of. the‘ m&_&@-1 siirface~‘;featu& reminiscent of condensed me&layers are olk+ne&~~ &&rn-$y; z_the”$&k-_ potentials at. high scan rates are shifted from their ~~equjlibrinm~ $_alues_‘~ The cathodic peaks are shifted towards more-~’negative _zg&II_ tiie~~iiikdi;i: ': peaks towards more positive potentials. This is evident fromi;Fig~-Y-~, where the cathodic and the. anodic peak’ potentials of: the- -different quinones in bilayer vesicles of phosphat_idyl-choline are presented asa func; -’ tion of pH for the scan rates of I V/S-~ V/s. At. slow scan ratesthereY. versible redox potentials are approached. There isn o signific~nt~~.d.iffe~~ C ence between PC and PS vesicles; in the entire range -of --pII’s ~betsveen,. 5 and IZ. In vesicles similar. to monolaycrs, undecyld.&ydroxyl+benzoquinone shows a steeper variation of the peak potentials _:vith:pH than-~ expected when one proton per electron is transferred_‘du~~~-th~lrediict~~;i-_-: process (Fig. 6). Iti this particular case,. the oxidatliolL-ana-i~tio~~~~-~ _ po$+i+_ : tentials depend on _the electric charge of the vesicle. The redo_& -.

..

E;ig.~ 5.

-

--- f-.~. i .: ,-

Fig_ 5_5::. pH dependence of then cathodic (0 e) and of the anodic (be)- p&&pot&tiaKof IO o/0 VK. in PS vesicles (upper set of- lines) and-in PC vesicles ; lipid ,con
272

Miller and Silverstein

of the dihydroxyquinone embedded in PC is more negative by about So mV than that in PS vesicles_ On the other hand di-te&-butyl-benzoquinone in vesicles produces a somewhat smaller variation of the peak potential (only about 50 mV) per pH unit. This decreased variation of the peak potential with pH is probably a result of the localization of this quinone in the middle of the bilayer. There the proton concentration is very low and part of the proton supply may come from the dissociation of water molecules.

Discussion

Vitamin I<, has a highly hydrophobic phytyl group containing twenty carbons attached to a naphthoquinone in the 3-position_ This enforces orientation of the compound in the surface be it in pure monolayers or when mixed with other lipids_ The orientation is pronounced enough to force the naphthoquinone residue which is not very hydrophylic, into the polar region of a condensed surface layer. This configuration inhibits the access of the naphthoquinone to the electrode surface contracting the monolayer from the hydrophobic si‘de ; consequently both the reduction and the oxydation potentials shifts away from the equilibrium redos potential. The shift in the potential of the protonrequiring reduction process is more pronounced than that of the o_xidation potential_ The 2.5 di-tert-butyi-r,q--benzoquinone has the relatively polar quinone residue embedded between the two hydrophobic butyl groups. This configuration facilitates the movement of this compound through the non polar part of the surface layer. Consequently the tendency toward irreversible reduction and osidation potentials in condensed monolayers or at high- vesicle concentrations is relatively weak and reversible pseudocapacitance peaks are almost always observed. .The undecyl-dihydroxyquinone is the most hydrophylic of the three quinones investigated_ Because of its finite water solubility it tends to escape from a condensed monolayer and its signal decreases with time- When incorporated into vesicles it may be distributed between the membrane and the aqueous phase. Nevertheless, in condensed monolayers (above the collapse pressure) and at high vesicle concentrations irreversible reduction peaks are observed_ However, even below the surface concentrations, when potential shifts due to irreversible processes are observed, another reversible peak appears_ This second peak could be- due to the oxireduction of a semiquinone or of a quinhydroneThe quinhydrone could be stabilized in a hydrophobic medium by hydrogen bonds between the hydrosyl groups_ Another puzzling phenomenon is the large dependence of the peak potentials on pH (about 95 mV/pH). This pH dependence of the pseudocapacitance peak potentials indicates that in the reduction process (equation II) a larger number of protons than of electrons is involved.

Redox

Kinetics

of

Quinones

in Lipid

Modolayers

27-3

Qm- + ne- + (n + -m) H+ ze QH
2.3 (n + m)-KT

uof-

pH

nP

-: : @I) __

. : : ^

The quinone can accept more protons than electrons durin~reduction. if at least some of the hydroxyf groups are ionized in- the quinone but snot in the hydroquinone. e-g_

OH f3i

L,

To explore this possibility RIrs SARAH. ROGOZINSKY of _the Biophysics Department carried out an acidimetric titration of the quinone incorporated in PC vesicles_ The titration curve is presented in Fig. 7. The pK’s of the first and the second phenolic groups are S.r and g-g respectively.

11 -

Fig.

I OS

, 1.0

NaOH(equivalent) r I 1.5 2.0

7_

P&eritiotietric titration curve:of UBQ(OH),. Full line measured, dashed line, corrected for. the number of (OH)ions in soluttin. zy_

The pK of the first phenolic group of the corresponding hydroyuinone isclose to 10. Binding of an extra proton during reduction offers an interesting possibility for coupling redox reaction with proton transfer and for building proton gradients.

Acknowledgement This research was supported by a grant from the Israel-Uk-Binational Science Foundation (No. 49634).

Miller

‘T-k

and

Silverstein

References 1_

\VILSOX and P-L. DUTTON. in EZecfvorz and Coupled Eqtergy Tvankfev ix T.E. K~;u~and AI. KLIXGENBERG. (Editors),Marcel Dekker and D-F. Wilson. New York (1971). p_ 221, J_ G. Lindsay-. P.L. Dutton (1972). Biochemistry 11. 1957 D_ \VALZ and O_ KEDEM. J_ Menzbvane Sci. in press A. KATCHALSKI- and P_ SPITNIET,J_ PoZyynz. Sci. 2, 43~ (1949) I<. J_ VETTER, Elecfvochenzical Kinetics Academic Press, Xew York (rg67) p_ 116, J_A_V. BUTLER, Trans. Faraday SOL 19, 729 (1924) T_ ERDEY GRUZ and &I_ VOLBIER. 2. PAYS_ Cheer. Abf. A 150, 230 (1930) R.E. PAGAKO and I.R. MILLER, J. Colioi~ZInferfnceSci. 45, 126 (rg73) 1-R. NILLER, Y_ RISHPOS and A_ TESESRXUM, Bioektvochenr. Bioenevg. 3, D-F_

BioZogicnZ Systems

2 3 4

52s

(1976)