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Spectroelectrochemical studies of stoichiometry, energetics, and kinetics of heme proteins: cytochrome c and cytochrome c oxidase
Bioelectrochemistry
and
Bioenevgetics
1, 389-406
(1974)
Spec troelectrochemical Studies of Stoichiometry, Energetics, and Kinetics. of Heme Proteins : Cytochrome c and Cytochkome c Oxidase * by THEODORE KU\VANA and WILLIAM R. Department Department (USA)
HEINEMAN
of Chemistry, Ohio State Universi+y, Columbus, Ohio 43210 ; of Chemistry. University of Cincinnati, Cincinnati, Ohio ‘45221
Two heme proteins, cytochrome c and cytochrome c oxidase, have been investigated in our laboratory by spectroelectrochemical techniques. In this paper we describe our spectroelectrochemical approach for the evaluation of stoichiometry, energetics and kinetics associated with each The spectroelectrochemical of these heme proteins and their mixtures. method involves the electrochemical generation of a mediator-titrant (either oxidant or .reductant) at an optically transparent electrode. The subsequent exchange of electrons between the mediator-titrant, and. the heme protein is monitored spectrophotometiically by light passing directly through the optically transparent electrode and ,the solution. The heme protein can be cycled conveniently between its reduced and oxidized forms. An accurate assessment of the number of. electrons (n value) transferred to or from the heme protein can be obtained from the charge required for titration. fl values for cytochrome c oxida%, cytochrome c, modified cytochrome’c and my6globin have been measured in this way. 0’ values, for cytochrome G and for.. the redox components of cytochrome c btidase have been evaluated from the shape. of absorbance-charge curves obtained by the spectrbelectrochemical method. Assignments are made on the basis of comparisons between experimental Potentials are reported *or curves and computer-calculated curves. cytochrome c oxidase in the presence and absence of cytochrome t% ‘The applicability of spectroelectrochemical methods for the measurement of ._ electron exchange with heme proteins is demonstrated. :
l Presented at the 2nd International Symposium on Bioelectrochemistry. Pont A Mousson, x-5 Oct. 1973
390
Kuwana
and Heineman
Introduction
Heme proteins are a significant aspect of the enzyme sequence in They are essentially redox species comoxidative phosphorylation. l posed of’ metal ions enclosed in a. nitrogen containing macrocyclic ring as well as a complicated biological framework. As such, they are amenable to study by electroanalytical techniques. Two of these enzymes, cytochrome c and cytochrome c oxidase, have been investigated in our laboratory by spectroelectrochemical technique and are the subject of this paper. Cytochrome c oxidtie is the terminal enzyme in the olddative phosphorylation chain and is responsible for the reduction of molecular 0, to H,O. It is thought to contain two Fe-hemes and two Cu atoms in the basic unit with a molecular weight of ca. ZOO,OOO(see refs. z-5 for reviews and discussion)_ Unfortunately, the structural relationship of these four entites is not known except .for inferences to their possible interactions from various chemical and physicochemical studies. The o,xidized oxidase can accept up to four electrons, and the generally accepted assumption is that the electrons are transferred to the metal centers with Fe being reduced from the +3 to +2 and the coppers from +z to +I oxidation states. There is no general agreement at present as to the similarity or dissimilarity between the two Fe-hemes or the two Cu in the o_xidase. Cytochrome c is the enzyme which transfer electrons to cyt&. chrome c oxidase. In contrast to the situation +h oxidase, the structure of cytochrome c in the crystalline phase is well characterized in both the oxidized and reduced states by the X-ray studies of DICKERSON and co-workers. 6 Some questions of signi&cance with these two heme proteins are as follows: I. the number of electrons transferrable to each protein (i.e. the n values) ; 3 the redox potentials, UO’ values ; 3: the interactions of cytochrome c with oxidase and the oxidase‘ This latter includes in particular the kinetics and sites of elecwith 0,. tron transfer. Mechanistic aspects are presently under intensive study. These mechanistic aspects have been widely studied by a variety of physicochemical methods both with the highly purified phospholipid-free o-xidase and the partially intact and submitochondrial phosphorylating’ particles. Cytochrome c oxidase is also s&r&cant in that this enzyme. not only catalyzes the O2 uptake, but also provides energy for the cell by coupling the energy ‘of the electron transfer through the cytochromes to the process of oxidative phosphorylation. The question of stoichiometry between cytochrome c and cytochrome c oxidase is not a trivial problem, and, in the literature (see ref. 2, pp. 7778, and ref. 5)) one can find differences in the expression for the stoichiometry between these two entities. In the area of energet&, there is considerable uncertainty as to, the redox potentials of
‘.
Energeticq
and -Kinetics
of, Heme
Pm&_:
” :g
‘.
.
.
:
_
all the possib!e :couples involving. .c@chrome
.--c.:, : ,o~~~e.~~ib’oth;j~,:lthe ;. purified and inthe, partially ,intact states.. And,:~~ough:‘:theie.,h~~~:.been:Iconsiderable kinetic _study. conducted;: the..,.~etails,ibfI,the.~.~~ti~tro~~~: transfer are still not :Iwell describedi” parh~uhrly g,wijzh ;,.@+eiA~:~.:,t~~i-$he T rkchan&n of 0, reduction.: Thus; there remain problemsr~~~~.~~.,~ be defined. ,+ electrochemical in nature. d?aJir$~~~~v?ith :-these::$wo.;.:heme :” :.: :i.y,54 .r:;.;.; 1 ,. c,., ,:_ ;“, :.-’ ::,. :.;‘7 ‘,. : ;t..f.: f .:.2\.,‘i:;,: ; :_. proteins; -., In
&is
,pa@r.’
we’
&all.,
&&be
I om.. ~s~~~~~~~h~~~.,~~~.~.,,
preach. 7,s. to .evaluate stoichiometry, entige&.~~and .-kineti& ,&.&&t,edL~ with -.each of, tl&& h&e protein& and th&, -mktures:- : ....:‘, .:,: ,::;.;;r.y..L :.‘-....,:_,.;f$ :_ .. . . . ...: : ‘i-,‘.... t‘. II;;, ,.. ,’ ., :::. :,
..
both
a
... ..
,::; .7:-‘T.-+g :' .: ..'1 :_.,:._ .j:;,,.__'.~._j‘<, .‘:_.,, OTE“&d---a 'pt' d&i&de,,.'&O @&iT:eg
redox level. of the solution can. be coikhi~hilj;‘ ;,_A&$:.r&Uy,_ @@t&!l using mediator-titrarits, while simultaneous +tentio&etric and optical’-. measurements can be taken,
‘C
‘
392
Kuwana
and
Heineman
employed, for example diaminodurene, pyocyanine, anthraquinone sultetramethyl-+phenyl-enediamine (TlMPD), fonate, ferri-ferrocyanide, phenazine methosulfate or ethosulfate. substituted bipyridylium salts (com.monLy called viologens or paraquats), various indophenols and These mediators, in order to produce a stable, meaningful pO_ others. tential must exchange electrons with the b&component and also with the electrode. Thus, the total level of electron exchange must be sufficiently high so that a poised potential is achieved. A poised condition may not abvays be achieved (i.e. reports of potential drifting l*J3), which is indicative of slow electron exchange with the indicator electrode. The alternative explanation , sloW attainment of equilibrium in solution, is less IikeIy unless a corresponding drift in spectral features is reported. -
red
t
u Fig. r. Schematic diagram of eIectron transfer electrode (OTE or Pt) to or from heme proteins. For example, mediafor--titraElt A may be MV’+ or MV+.. mediator-titrant B may be ferricyanide-feocyanide, or O,-HIO. Heme protein A may be cytochrome c and B cytochrome c oxidase or others. The applied potential, U, governs the direction of the electron transfer.
OTE + Pt
Depending on the electrochemical characteristics and the value of the UO’, mediators also serve as cozclotnetric titrants in our studies. The two uses appear almost exclusive of one another. That is, for a mediator, the U”’ values between the mediator and the bio-component should be close together whereas, as a titrant, they should be far apart for completion of the desired reaction (equilibrium lies far to the right). The best situation is where one has a variety of mediator-titrants with UO’ values graded in steps of a IOO mV, or so, tbrough a range of for example -500 to +Soo mV. Then, two or more of these mediator-titrants can be utilized to esamine a particular b&component. From the change in optical absorbance of the b&component vs. the electrochemical change, accurate assessment of the equilibrium and hence of both n and UO’ of the b&component is possible, assuming the n value and UO’ of the mediators are accurately known. Unfortunately. our arsenal of electrochemically well characterized mediator-titrants is still limited. For
Energetics
and
Kinetics
of
Heme
Proteins
393
reductions, we have evaluated a series of viologens l4 which undergo a stepwise, one electron each (EE mechanism) electron transfer from the Eation to the radical monocation and then ,to the neutral species. The values for the two steps are sufficiently’ separated that -the radical can be .preferentially generated as the reductant. The UO’ .vab.res-range from -500 to -300 mV. For oxidations [Fe (CN)J3- has been, generated from [Fe (CN),]+ (UO’ = 424 mV). Viologens and the Fe-cyanides are nearly ideal mediator-titrants ;- it = I, good chemical stability, fast electron exchange with most components’and with the electrode, and low optical absorbance in most of the spectral regions where heme proteins absorb. TMPD (UO’ = 270 mV) has been widely ,used, although it does not have a simple two species equilibrium at pH 7. Molecular 0,, the physiological oxidant for cytochrome c oxidase, has been coulometrically generated at both the Pt and the SnO, electrode as a titrant in some of the studies reported here. Cytochrome c oxidase was isolated from beef heart mitochondria and purified according to C. R. HARTZELL. The heme a and Cu content were 14 pmol/g protein and 15 pmol/g protein, respectively. Horse heart cytochrome c was prepared according to MARGOLIASH.rs All reagents were high purity, biological grade. Spectra have been taken, usually at X-Y recording speeds, using an improved version of an earlier described Is dual beam, direct log absorbance readout, rapid scanning spectrophotometer [(Harrick Scientific Corp., Ossining (N.Y.)]. All potentials are corrected and reported, with respect to normal hydrogen electrode (N.H.E.).
StoicAiomed7y As a prerequisite to evaluation of the stoichiometry between bioelectron transfer components and to understanding the energetics and kinetics of reactions involving biocomponents, it is desirable to have an accurate assessment of the total number of electrons (12 value) transferable to individual components. Accurate and precise values for n .have been measured by the indirect coulometric titration method. Fig. 2 A shows the absorbance--charge (AA-AQ) plot for the reductive, followed by the oxidative, titration of cytochrome c odxidase. The methyl viologen radical cation, MV+ , was electrogenerated from MVZ+ (Uo’ =.~#6 mV) for the reduction and 02 was clectrogenerated at the Pt microelectrode .‘. for the oxidation. The fi value can-be calculated from the total charge or equi+lents required to titrate’ all of the oxidase or from the slope of the A:A-AQ plot according to equation I:
Kuwana and Heineman
394
where AE is the difference molar absorptivity (reduced - oxidized), in f&I-lcm- 1, b is the optical path length of the cell in cm, T’ is the. cell volume in Liter, and 91tis the slope in a.u./eq. Results for several heme proteins titrated coulometricahy are summarized in Table I. Since n values were known already for these heme proteins, the data principally serves to illustrate the precision and accuracy attainable by this coulometric method_ In some instances, the reductive oxidative titration was repeated through several cycles with very little variation in the resulting n value escept for cytochrome c oxidase which required about IO Ojo more charge during the initial reductive titration. The reason for this is presently not known.
Table
I.
CouIometrically
determined
n values
72
Component
Value -
I ( kidation
Reduction
a. Reductions (C%J3--* phosphate 6. 30.6 t.
52-7
d. 16-7 avg.
I.OZfI 0.04 I.19 r_I4+0.08 2_15+0.3 j
by electrogenerated except oxidase by 0,. buffer at pH 7.0.
~_LLM myoglobin, p&f modified to 26.7 @f 6 solutions.
e. 5.5 PM cytochrome
0.5
M
MV+; All
Ka[Fe(CN)
cytochrome cytochrome c oxidase,
0.03 I-04 1.03 & 0.09 2.04+ 0. ro
o_gg&
t, 0.5 c, 0.5 0.6 mM
fi.
7
‘,*
Icl Xeduction -_
.-
Myoglobin b Modified cytochrome GC Cytochrome cd Cytochrome c oxidase 6
heme.proteins
of some
r.oo+ 0.01 o-97 r.oz~o.14 r.g8+_0.21
2 3xidation
2
I.OZ+-0.01 0.95
r.o4+o_os 2..14+ 0.24
oxidations by electrogenerated [Fe solutions contain 0.1 M N&l, 0.1 M J, avg.
m&f mM
3 solutions.
MVzf, MVzf,
0.5 m&f [K,Fe(CN) 0.5
to 2 mM
J_
KdFe(CK)J,
MV”-+-, 0.1 O/e Tween 20, avg. 4 or more
solutions.
The n values measured by indirect. coulometric titration represent the total number of electron-equivalents consumed by the heme protein per mole of the optically monitoied species. In contrast, the it value measured by potentiometric titration is specific for the specks whose spectral band is used for calculation of the ratio of reduced to oxidized forms. Consequently, n values which are obtained by the coulometric method may differ from those obtained by the potentiometric method. In the. case of cytochrome c oxidase, a value of 2 e- per heme a (one Cu per heme is titrated simultaneously) is obtained by the’ spectroelec-
Energetics and Kinetics of.,Heme Proteins
39.5
trochemicai coulometry whereas a I e- value is obtained by the potentiometric method I7 since it is only the heme n constituent whose ratio of redox forms is being monitored at 605 nm.’ The indirect coulometric titration has several advantages over conventional chemical titration procedures for measuring n values. These are: I. solution conditions remain essentially invariant during the titration ; 2. smaLl increments of the titrant (charge) can be added accurately and conveniently at any repetitive rate ; _?. the bio-component can be repetitively cycled through various oxidake or reductive levek rapidly (few minutes, if necessary). The supply of titrant is infinite since its original redox state is regenerated by electron transfer with the b&component. athough the electrical potential applied to the electrode is thermodynamically sufficient to cause direct electron-. transfer to or from the bio-components, the overpotential for transfer is such that the electron is transferred for all practical purposes quantitatively through the mediator-titrant. -.
Energetics
.-
The potentiometric method has been employed extensively to determine UO’ values of both puri@ed and intact components of the oxiThe concentrations of the components dative phosphorylation chain. have been assessed spectrophotometrically during titration and n and UO! values, ‘evaluated from. the familiar nernstian plots of Urn-~ ,vs. the appropriate logarithm of concentration ratio (expressed in optical absorban’ce) . UO’. values of heme proteins can also be measured by evaluating solution equilibria between the b&components and another redox component- whose UO’ ,is accurately : known by independent measurement. Assuming a quantitative equilibrium transfer of charge from :the titrant to the bio-component, it is possible to evaluate the energetics (UO’) of a bio-component from analysis of the AA-AQ curve cbtained from the The applicability of this method was indirect coulometric method. demonstrated by ,HAWKRIDGE 7 for cytochrome c, where the mediatortitrant system of methyl viologen (UO’ =‘-446 mV ZIS. N.H.E.) and The, UO’ ferri-ferrocyanide ( UO’ = 424 mV OS. N.H.E.) was employed. f6r cytochrome c was evaluated as 258 & 17 mV ZLS.N.H.E. In ordTr to obtain accurate values by this method, the UO’ of the reference niediator (ferri-ferrocyanide in this case) sliould be within ca. 200 mV of the bio-component’s UO’. The case of cytochrome -c’ oxidase is complicated by the presence’ of four redox centers per molecule, two hemes and, two Cu. Information concerning the redox potentials of these four components of cytochrome c can be inferred from the experimental AA-AQ curve shown in Fig. z A. In this titration the UO’ .of the two titrants are sufficiently removed from. those ,of the bio-componentS that their influence. on the AA-AQ
Kuwana and Heineman
396
Fig. 2. o-6 m&f MV+. A. hAmor AQ plot of coulometric titration of s-5 g&f cytoclqome G oxid-. 0.1 M NaCl. and 0.1 O/O Tween 20 in 0.3 M phosphate buffer pH 7-o. No correction for IT+ sidual charge. (0) Reduction by PUN+: (0) Otidationby 0,.
&l(05-AQ plots for cytochrome G oxidase. (-) UaraL - UO’cUL or U”‘_+= UO*cUII = U”‘ar= U”‘cU,; (- -) Cl”‘+ =
B. Computer simuIated ua*cLla
-
UO’CU
L
--
280
; (--+-)
Ua'+
=
350,
ZYa~
=
U”‘Q~
=
210,
UJ’aH = U”‘CU~~. 350,
U”‘Q~
U”‘aL
=
=
2.10.
310.
curve is negligible. Only two situations will give a straight-line plot of AA-AQ as observed in Fig. 2 A : I. the standard redox potentials of all four components are ider+cal; 2. the heme potentiafs are separated with each of the Cu potentials separated identically with the hemes. The AA-A0 curves for this multiredox heme protein can be computer simulated “using the four Nemst equations : U =
P’heme uH-
59
log([heme
a,,(red)] /meme
u =
U”‘heme aL -
59
log(@eme
+(red)] /[heme aL(ow
(3)
u =
Uo’cy,
I[CuH(ox)31
(4)
-
u = SO’c uL-
59 logKCu&ed)l
59 log~CCu,(red)]/CCu,(ox)])
4I(om
(4
(5)
As the redo-u potential, U, reflecting the redox state of the solution, is var-
ied from a value in which all the components are in their oxidized form to a value in which they are in their reduced form, the resulting ratios of each
component (each heme and Cu) can be computed in terms of the consumed charge and of the change in the optical absorbance at 605 nm. Results
of such simulations are shown in Fig. z B where UO’ values of 350 and
Energetics
and
Kinetics
of Heme
Proteins
397
210 mV were assigned to the two hemes and the values of the two copper potentials were varied as indicated in the legend. It is estimated that a difference Of a u”‘Cu from a u”‘h eme a by IO mV, or more, should be experimentally detectable by the deviation from linearity of the AA-AQ curve. Deviations from linearity of the type noted in Fig. 2 B have not been observed (i.e. plots reported by KUWANA 8 and VAN GELDER lS are linear in the intermediate region of titration). It should be noted that the reported values of the Uo’cu based on the behavior of the S30 nm band would definitely produce deviation from linearity in these plots. In Fig. 3 the AA-AQ is plotted for the indirect coulometric titration of cytochrome G oxidase in the presence of MV2+ and ferri-ferrocyanide. lg In this case Uo’ values for the redox components of cytochrome c o-xidase can be determined from the deviation from linearity caused by equilibrium with the ferri-ferrocyanide couple. The solid line is for a computer simulation of the AA-AQ using the UO’ values of 434, 350, 350, zzo, and 220 mV for ferri-ferrocyanide, heme aH, Cu,, heme aL, and Cu,, respectively. The calculation is corrected for the weak chargetransfer complex 2O formed between [Fe (CN),]+ and MV2+ dication. The effect of varying the Uo’ value of Cu, is illustrated in the same figure. The agreement between the calculated and experimental results is excellent for the values of UO’ being 350 and zzo mV for the two heme a-Cu pairs. The same experiment was repeated with TMPD replacing ferri-ferrocyanide as a mediator. Is The results appear to be identical within experimental error.
0.20 -
0
h
-s 0.16
is ”
-
: 0.12
-
0.08
-
o_oc
-
0.00
-,
g 9
.i ” 1 00
I 40
0
\
Qneq/cm3
I 120
I 200
Ii0
Fig. 3. AA-AQ plot for reductive MYa+, 0.5 mM [Fe(CN)d]s-,
coulometric titration of 9.3 FM cytochrome c oxidase. 0.5 m&I and 0.1 o/o Tween 20 in 0.3 M phosphate buffer pH 7.0. Corrected
for any background charge.
A. Computer simulated for UO’+ =
U”‘C~, =
(0) experimental points. B. Computer simulated for U”‘+
350, UO’cCH=
=
350 mV, 280.
U”IaL= U"*+
=
UO’cUL= U”‘cUL =
220
220
mV. mV.
Kuwans
39s
0
2
4
and
Heineman
6
Titration of 17-5 @Z cytochrome c and 6.3 PM cytochrome c oxidase by reduction with MY+-. Solution conditions same as in Fig. Z. [Upper left hand comer insert : AA-AQ plotsfollowing Main figure : y 605 and 550 nm absorbance bands ; charge corrected for background]. 0 experimental points. Solid .=IA45501(r~~ % -AA,,,)]. x = kL4,,/(100 % --A&,,)]. lines simulated for CT”‘+, 1: 350 mV, U”‘+ = zoo. 210, and 220 mV. UO’w~, c=250
mV.
_
Uo’ values of the two heme a components of cytochrome c oAridLe have also been determined from titrations of mixtdtes of cytochrome c and cytochrome c o_xidase. The AA-AQ curve (monitored at 605 and 550 nm) for such a titration is shown in Fig. 4 (insert). It can be concluded from the shape of the absorbance change at 605 nm that the ZP’ of cytochrome c is intermediate of the UO”S of the two heme a’s. The
Energetics and
of ,Henih
.Kiiieiiics
Proteins
399
_
behavior of the-.ofitically monitoredhcmes can -be separated from. theCu% (which also consume charge) by .pl&ing I:the.: logarithms:.of : the: titios' :ofa the reduced to. oxidized -forms as sho&n~,incFig.. 4; :-The-d&a are plotted as., log[c(red)/c(ox)] vs;.:‘,log[~~,(red(/a&(ox)] : i w h ere : ‘+&)~c(ox)~.-+ = ‘AA&/ g&(red) /I% .&, AA dljs /(qjo.-o/d~~ (IO0 70 --AA_& .. -and -AA aas). AA representing the percentage of the total absorbance changer The ( &A.lting sigmoid-shaped cur& : is characteristic :‘of ,::-a: ~‘uiultiredox system_. .,’ I :. _. ., : .. ...,, 1 ., _ : ,..:‘..:~I\‘:. .:.:,-;-!..._:;_:c:i,_i ” Similar. curves can be obtained: by calculating -the log terrhsof .the .. fohowing Nernst -equations,- as the potential .U of- theasystem’: (solution) 2; ;, ! is varied : :
u =
UQ’cgt
j.
u=
UQLne~
u
=
c
59 log[c(red) /c(cox)]- -.J’
-
-
aL -
cJfJr,,
59
59
Jogpeme
.
(6)
zz,(red) /heme a,(ox)]
Iog@eme &(=e&/heme
(4
%(0x)]
(3) ,
where the -subscripts H -and L ‘&fei- to high. -and .loii;. potential :hem&., Log[b(red) /c(o)r)] ‘is then_ plotted against log([&(red)- $L &@l)] /[aL(o,x)“q. assigning +a++ -t %&m)--me solid. Curvg- in Fig. 4 were obtained':by of, W&r = 250 mV, vT’hcmc+ = 35o ‘mV’: and’ varying -the.;.value of that’ ‘th,e cu.+ :are @te U?.‘hall= 5. from zoo ‘to 220 mV_ : _Jt is ,ap&+nt sensitive. to- changes in U”‘. The .U?’ valuesof ra, and e&re &z&ned_ 35o and ZIO .mV; respectively;: based
‘.
m+-2 . MV+-
+
+_,,e~.&-_~+*
.‘I
cytochrome ca
kf,-
*
kb ':
.
.__I. I.
MV+*..+
-‘. i
‘.- -._ I.‘. ,,,__1: i.r-,iy]’ : 1.
cytochrome c&
.:.:. ,-(8)
_._: .: -....! i .: ;,I ;, >,c ;. _:;.:-:,;_:'L_,..: by,the.,~m~
\t&' c&Z the electrogenerated ‘kV+ : is '~&&d.&& dizzed cytochrome c. The rate constant,. kf, for the homogen&%rs.- e:
where
Kuwana
400
and
Heineman
transfer can be conveniently evaluated spectroelectrochemically by optically monitoring at set wavelength for either NV+or reduced cytochrome c as a function of time during a chronoamperometric experiment. Spectrochemical evaluation of rate constants for such mechanisms was demonstrated earlier and extended to an enzymatic electron transfer by ITO. 2z A discussion of the concentration-distance-time (c-x-t) and absorbance-rate constant-time (A-k-t) profiles which were computer calculated by the method of digital simulation, 23 will be. presented for the purpose of cIarifying the spectroelectrochemical approach. In a generalized EC catalytic reaction scheme,
where A* is electrogenerated at a diffusion controlled rate, the c-x profiles as a function of time, t, are shown in Fig. 5_ The initial conditions of the esperiment can be tied : for this simulation, at t = o, the concentration of A was set equal to 2. The concentration of A* depends on the diffusion coefficient of A, the concentration of 2 and the value of kf, assuming kf+ kb. Explicit in the mechanism is the high overpotential for the heterogeneous electron transfer to 2 so that it reacts at the electrode at a negligible rate compared to A. This condition ap-
-c e-
A-A* Li__________*
A-+2
-K-A
+
27:
k =lO’ 1.0
-----A2
P
E
Y
r”
0
ms
s
0 Distance,
cm
Fig- 5_ Concenbation+liscance profiles for species, A, Af * Z and 2’ as a function of t -0, I ms, 4 ms and S ms for second order EC catalytic mechanism. Rate constant k = IO' ; at t I o, [A ] =
[Z] =
x0-’
M_
Energetics-and Kinetics of I Heme Proteins +=+A%--,
_-.._-. . = ?OC
&=O,Y.~
“.~__-“_‘__“
A-+Z&i\+Zz
_ .rr-6
401.
:
,Af.
.;y
.. . .
:‘.
‘.,..
ms 0
1
2
4
3
‘_
..
._ Fig. 6. Absorbance as a function of time cokpnted for, varioi v&es of ,k for second ord& Ed: &atalytic mechanism during potential step (chronoamperom&ic) expbiment. !$ecie& k* : a 1.04. At L= O, [A] = [Z] = x0-8 M ; DA = 101 cm* s-1. CA* 0pticaUy monitored. ..
to be fulfilled f or many of the nalxked hem& proteins; The ~o&cal absorbance, A, changes during the electrolykis. ,tjrqe. depending on kf atid plots of A - t for various values of kf are’ shown in Fig; .6.., Fck ltige values of kf and/or small values of molar’absorptivity of the &onitored species, it may be necessary to signal average’ the-optic+ respok during’ repetitive electrochemical pulses: or utilize internal reflection techni@s.
pears
Fig. 7.
_. .. *; *_
:
b+
..
‘.
Change in opticalabsorkance~+3 a fm$ion of time during cqu~pav~i .p&+ti$ pulse. A. Monitoring at 630 nmthe for7 mation and removal of &IV+-: B: In the presence of cytcuzhrome c. Absarhance is signala==@ (135op*) =+outfmm Puke Iength 8 PAR Wav.efo+ Ednctor.
-
Time -_)
ms, io&
sqnareywav~ &,&tion’of,d$
630
nm
Dm+z
repeated_ every &_--&fi’&$&&
=
D~c-r.r~10*~?s-‘(ref.25)r
6.86 & xd4
‘2 s_fot‘
&in- ‘ii+’
403
Knwana
and
Heineman
Preliminary results 24 of the A ,- t patterns for the reaction of &IV+* with oLxidized cytochrome c are shown in Fig. 7. Curve A is the signal averaged optical response for monitoring &IV+- during a 8 ms square wave electrochemical pulse which was repeated every z seconds for a total duration of 45 minutes (1350 pulses). The signal was averaged using a PAR \V.AVEFORM EDUCTOR (100 channels, maximum time resohrtion APPLIED RESEARCH, Princeton, N-J.). I ps per channel ; PRIXETOX In curve B of the s+me figure, the experiment is repeated with equal moIar concentration of cytochrome c~ present. The rising portion of the absorbance (8 ms duration) corresponds to when the U,,U~ is a value such that MV** is being produced at a diffusion controlled rate ; it reacts with the oxidized c as it moves toward the bulk solution phase. When Uapp~d is stepped back to a value such- that reaction (7) is reversed, the absorbance decreases with time. The Kf of reaction (S) can be analyzed from both the forvard step time (8 ms), or from the decreasing portion of the absorbance. Only the former will be discussed here. The normalized absorbance (A -=B/A-~A) is computer-calculated for various values of kf and t, using the initial concentrations of &IV*: and cytochrome cO~~ and the known diffusion coefficients of the tivo species. By fitting the experimentahy determined normalized absorbance to the computer-simulated normalized absorbance, ‘the value of kf Ca.c be’obtained. For the conditions of the esperiment (?ee Fig. 7). the rate constant was evaluated as equal to 5 (-& z) x .IO"I moF s+. This value is an order of magnitude larger than the rate constant for the cytochrome cdUc~ - cytochrome c,a self-exchange and an order of magnitude less than the rate of [Fe (CN),] 3--o_xidation of cytochrome ctdUd. Because the driving force of reaction (S) is large (AGO’ = -16 kcal mol-r as estimated from the UO’ values of the two redox couples) the rate is espected to approach diffusional rates except for any steric factor involving the reactants. SUTIN, 28 assuming transfer through the exposed edge of the prophin ring, estimated that this electron transfer site occupied about 3 o/0of the total surface area of cytochrome c. Assuming a collisional model and a 3 o/0steric factor, the estimated rate is on the order of 108 Z mol-1 s-l_ Obviously, the measured rate is considerably less than this. To quote SUTIN.~~ “ ...mechanism of electron-transfer to and from cytochrome c is a challenging problem and further studies using a wide variety of oJxidizing and reducing agents are necessary before any mechanistic assignment can be made with confidence “. Further spectroelectrochemical measurements of kinetic parameters involving mediator-titrants with cytochrome c and cytochrome c ,oxidti& are in progress. Discussion
For the purifieP cytochrome c oxidase, there has been considerable discussion regarding the similarity or dissimilarity between the two hemes. Also, there has been suggestion that cytochrome c oxidase titrates
Energetics and Kinetics of Heme Rote&
403
as one component (all UO’ values identical) in the absence of cytochrome c and that the presence of c or ferricyanide-ferrocyanide splits the Uo’ values of the two hemes. We would like to make .the following statements with regard to these two points : I. In a reductive potentiometric titration, both hemes will titrate as one component when a strong reductant is used. If a mediator with UO’ more negative by IOO mV or more (e.g. phenazine methoslilfate) is used, the hemes will still titrate as ooze optical component. The reason for this is that the two hemes are not well differentiated optically. Only if one or both Cu titrate at a potential sufficiently apart from the heme potentials, will there be a curvature in the AA-AQ plots (e.g. Fig. z B). 2. Because the two hemes titrate at different potentials in the presence of cytochrome c, or other mediators, doe-s not necessarily mean that these two hemes cannot be identical in the fully oxidized or fully reduced states. A possible reason for UO’ differences observed experimentally is that each electron transferred alters the charge and the electron density distribution, which consequently affects the GO’ for the next electron transferred. As suggested by several workers, 27*2athere appears to be evidence for heme-heme interaction. If so, it is expected that the potentials for the half-reaction involving the second heme titrated will be different from the UO’ of the half-reaction of the f&t heme titrated, even if the hemes were initially identical in the fully reduced or oxidized states. Interpretation as to the heme-heme interactions from potential data is tenuous, however, since equilibration between the hemes may occur through electron exchange with a mediator, or mediators (in case of potentiometric titrations), and not through direct electron exchange between the hemes. There is little information available on the latter. The stoichiometry and energetics correlated to tbe available kinetic data strongly suggests that the addition or removal of electrons occurs stepwise in two I.1 electron pairs. Each pair of electrons corresponds to a heme-Cu unit. From kinetic studies, 29 it appears that two electrons are added to the fully oxidized oxidase initially at a rapid rate. Thus, the rates of transfer to and from oxidase for each. pair (not to imply simultaneity of the two electrons transferred) may be different. If equilibrium titrations are performed on the time scale of several minutes to hours, what may be observed are the electrons filling or emptying cytochrome c oxidase according to the thermodynamics of the,components with respect to each other. However, if transfers are made on a much shorter time scale, kinetic differentiations may appear with respect to the site, or sites, of electron transfer. The problem is further complicated by the possible dependence of the mechanism of transfer on the type of reactant. Continued studies are certainly warranted and needed. The assignment of Uo’ value to the hemes and Cu’s is summarized in Table 2. Selected literature values are included for comparison purposes. It should be noted that the heme value of ca. 280 mV reported for the situation where the hemes are supposedly identical is an average of the Uo’ values of heme a= and heme q. Similarly, the potential for
Comparison
cyt a,
cyt a
2W.5) 225 0.0) I
as single component
designated
b. Frequently
c, Titrated
1.0)
225 (N I) 232 (- I) 55 W)
designated
ao,a1
la
35o&5 (1.0) 3503- IO( I .o)
high b ________-__
c 375 (1.0)
.364 (N I) 347 (N I) 251 (N I)
335-36oW)
280(x 80) c
(LO) 30( LO)
zoo-280(
2202
zrok5
10~ a
____
heme a
30(1.0)
3503- IO(I,O)
c oxidase.
Conditions
--.--_
highly purified partially purified
5 x 10-a M [Fe(CN) J85 x 10-4 to 10-a M [Pe(CN)JOo to 10-6 M fFa(CN) J*-
cyt c absent. cyt c present
cyt c present [l?e(CN)Js’ present
-------
e. Based on 830 nm band and EPR
d. Based on 830 nm band
220&
low
of U0’ values for purified cytochromc
a. Frequently
WILSON
CUSANOVICH
VAN GELDER l*
This paper
Source
Table z,
Energetics and
Kinetics
of Heme
Proteins
405
Cu determined from monitoring the 830 nm band appears to be near the average of the UO’ for Cu, and Cu,. Preliminary spectroelectrochemical coulometry at the 830 nm band shows close parallelism to the 605 nm band of the hemes.
AcknowIedgements
This investigation was supported by PHS Research Grant GM 19x81 and initiated during tenure of T. K. (NIH Special Research Fellowship I F03 GM 48486) at the Institute for Enzyme Research at the University of Wisconsin, Madison, under Professor H. BEINERT. W. R. H. acknowledges support from RESEARCH CORPORATION,COTTREL Grant 6720. The authors satefully acknowledge discussions with C. R. HARTZELL. Cytochrome c and cytocbrome c oxidase were provided by C. R. H. Data provided by L. MACKEY and M. FUJIHARA are acknowledged. The authors appreciate preprint copy of paper by CUSANOVICH and WHARTON.
1 2 3 4 6
6
7 8
8
10 11 12 18 14
A. LEHNINGER.
BiochenzisCry,
Worth
Publishers
M.R. LEMBERG. Physiol. Rev. 49, 48 (1969) T.E. KING, H.S. MASON and M. MORRISON, Systems, Wiley, New York (1965)
(1970)
Ed. Oxiduse and Related Redox
K. OKUNIJKI, M.D. KAMEX abd I. SEKUZU. Eds. Sfrucfure and Function of Cyfochromes, University Park Press, Baltimore (x968) E.C. SLATER, B.F. VAN GELDER and K. MINNAERT, in Oxidase and ReZafed Rsdox Systems, T.E. KING, H.S. MASON and M. MORRISON, Eds., Wiley. . New York (1965) T. TAXANO, R_ SWANSON, 0-B. KALLAI and R.E. DICKERSON, in Cold Spring Nor&or Symposia on Quantifaotiue biology, vol. 36. Cold Spring Harbor Lab-
oratory (1972) F-M_ HAWKRIDGE
and T. KUWANA, Anal. Chem., 45, IOZI (1973) W.R. HEINEMAN, T. KUWANA and CR. HARTZELL, Biochem. Biopliys. Res. Commun. 49, I (1972) ; ibid. 50, 892 (rg73) N. WINOGRAD and T. KUWANA, in EZecfraanaZyticaZ Chemistry, A Bard, Ed., Marcel Dekker, New York, in press vol. 7 J_W. STRO JEK and T; KUWANA, J. EZectoanaZ. Chem. InterfaciaZ JTkctrochsm. 15, 471
(1968)
A.H. C&WELL and B.C. PRESSMAN, Arch. Biochem. Siophys. 125, 318 (1968) M.A. CUSANOVICH and D.C. WHARTON private communication M. ERECINSXA. D.F. WILSON. Y. MUKAI and 33. CHANCE, Biochem. Res. Commun. 41, 386 (1970) E. STECKHAN and T. KUWANA, unpublished results
Siophys.
Kuwana
405 15 16 I7 18
19 20 21 22
23
24 25 26 27
28 29
31
Heineman
E. MARGOLI~~H and A. SCHEJTER. Ada. Profei= Chem. 21, 114 (1966) J.W. STROJEIC, G. GRUVER and T. KUWANA, AnaZ. Chem. 41, 4Sr (1969) P.L. DU~ON, D-F. WILSON and C_ LEE. Biochemistry 9, 5077 (1970) A-0. MUIJSERS, R.H. TIESJEMA, R.W. HENDERSON and B.F. VXN GELDER, Biochim. Biophys. Acfa 267, 2x6 (1972) L.N. K~CKEY, T. KIJW_~X_~ and C-R. HXRTZELL, FEBS Letf. in press A. NAKAHARA and J-H. W-G. J_ Phys. Chem. 67, 496 (1963) K. MINNAERT, Biochim. Biophys. Acla 110, 42 (1965) M_ ITO and T. KTJWANA, J. EZecfroanaZ. Chem. Infevfacial Elecfrochem. 32, 415 (1971) SW. FELDBERG,
in EZecfroanaZyylicaZ Chemisfry, vol. 3. -4-J.Bard, ed.,LMarceI Dekker, N.Y. (1969) Data provided by M. FUJIEARA, Ohio State University A. EHRENBERG, Acta Chem. &and. 11, 1237 (1937) N. SUTLN, Chem. Brif. 8, 148 (1972) D.F. WILSON,. J.G. LINDSAY and E.S. BROCKLEHURST. Biochim. Biofihys. Acta 256, 277 (1972) D.F. WILSON and J.S. LEIGH, Jr., Arch. Biochem. Biophys. 150, 154 (1972) Q.H. GIBSON, C. GREENWOOD. D-C. WHARTON and G. PALMER, J. BioZ. Chem.
30
and
240,
SSS (1965)
D.F. \VILSON, P.L. DunOI<, M. ERECINSKA, J.G. LINDS_%Y and N. SATO, Accounts Chem. Res. 5, 234 (1972) T. Tsrrirw~r and D.F. WILSON, drch_ Biochem. Biophys. 145, 149 (1971)
and
Bioenevgetics
1, 389-406
(1974)
Spec troelectrochemical Studies of Stoichiometry, Energetics, and Kinetics. of Heme Proteins : Cytochrome c and Cytochkome c Oxidase * by THEODORE KU\VANA and WILLIAM R. Department Department (USA)
HEINEMAN
of Chemistry, Ohio State Universi+y, Columbus, Ohio 43210 ; of Chemistry. University of Cincinnati, Cincinnati, Ohio ‘45221
Two heme proteins, cytochrome c and cytochrome c oxidase, have been investigated in our laboratory by spectroelectrochemical techniques. In this paper we describe our spectroelectrochemical approach for the evaluation of stoichiometry, energetics and kinetics associated with each The spectroelectrochemical of these heme proteins and their mixtures. method involves the electrochemical generation of a mediator-titrant (either oxidant or .reductant) at an optically transparent electrode. The subsequent exchange of electrons between the mediator-titrant, and. the heme protein is monitored spectrophotometiically by light passing directly through the optically transparent electrode and ,the solution. The heme protein can be cycled conveniently between its reduced and oxidized forms. An accurate assessment of the number of. electrons (n value) transferred to or from the heme protein can be obtained from the charge required for titration. fl values for cytochrome c oxida%, cytochrome c, modified cytochrome’c and my6globin have been measured in this way. 0’ values, for cytochrome G and for.. the redox components of cytochrome c btidase have been evaluated from the shape. of absorbance-charge curves obtained by the spectrbelectrochemical method. Assignments are made on the basis of comparisons between experimental Potentials are reported *or curves and computer-calculated curves. cytochrome c oxidase in the presence and absence of cytochrome t% ‘The applicability of spectroelectrochemical methods for the measurement of ._ electron exchange with heme proteins is demonstrated. :
l Presented at the 2nd International Symposium on Bioelectrochemistry. Pont A Mousson, x-5 Oct. 1973
390
Kuwana
and Heineman
Introduction
Heme proteins are a significant aspect of the enzyme sequence in They are essentially redox species comoxidative phosphorylation. l posed of’ metal ions enclosed in a. nitrogen containing macrocyclic ring as well as a complicated biological framework. As such, they are amenable to study by electroanalytical techniques. Two of these enzymes, cytochrome c and cytochrome c oxidase, have been investigated in our laboratory by spectroelectrochemical technique and are the subject of this paper. Cytochrome c oxidtie is the terminal enzyme in the olddative phosphorylation chain and is responsible for the reduction of molecular 0, to H,O. It is thought to contain two Fe-hemes and two Cu atoms in the basic unit with a molecular weight of ca. ZOO,OOO(see refs. z-5 for reviews and discussion)_ Unfortunately, the structural relationship of these four entites is not known except .for inferences to their possible interactions from various chemical and physicochemical studies. The o,xidized oxidase can accept up to four electrons, and the generally accepted assumption is that the electrons are transferred to the metal centers with Fe being reduced from the +3 to +2 and the coppers from +z to +I oxidation states. There is no general agreement at present as to the similarity or dissimilarity between the two Fe-hemes or the two Cu in the o_xidase. Cytochrome c is the enzyme which transfer electrons to cyt&. chrome c oxidase. In contrast to the situation +h oxidase, the structure of cytochrome c in the crystalline phase is well characterized in both the oxidized and reduced states by the X-ray studies of DICKERSON and co-workers. 6 Some questions of signi&cance with these two heme proteins are as follows: I. the number of electrons transferrable to each protein (i.e. the n values) ; 3 the redox potentials, UO’ values ; 3: the interactions of cytochrome c with oxidase and the oxidase‘ This latter includes in particular the kinetics and sites of elecwith 0,. tron transfer. Mechanistic aspects are presently under intensive study. These mechanistic aspects have been widely studied by a variety of physicochemical methods both with the highly purified phospholipid-free o-xidase and the partially intact and submitochondrial phosphorylating’ particles. Cytochrome c oxidase is also s&r&cant in that this enzyme. not only catalyzes the O2 uptake, but also provides energy for the cell by coupling the energy ‘of the electron transfer through the cytochromes to the process of oxidative phosphorylation. The question of stoichiometry between cytochrome c and cytochrome c oxidase is not a trivial problem, and, in the literature (see ref. 2, pp. 7778, and ref. 5)) one can find differences in the expression for the stoichiometry between these two entities. In the area of energet&, there is considerable uncertainty as to, the redox potentials of
‘.
Energeticq
and -Kinetics
of, Heme
Pm&_:
” :g
‘.
.
.
:
_
all the possib!e :couples involving. .c@chrome
.--c.:, : ,o~~~e.~~ib’oth;j~,:lthe ;. purified and inthe, partially ,intact states.. And,:~~ough:‘:theie.,h~~~:.been:Iconsiderable kinetic _study. conducted;: the..,.~etails,ibfI,the.~.~~ti~tro~~~: transfer are still not :Iwell describedi” parh~uhrly g,wijzh ;,.@+eiA~:~.:,t~~i-$he T rkchan&n of 0, reduction.: Thus; there remain problemsr~~~~.~~.,~ be defined. ,+ electrochemical in nature. d?aJir$~~~~v?ith :-these::$wo.;.:heme :” :.: :i.y,54 .r:;.;.; 1 ,. c,., ,:_ ;“, :.-’ ::,. :.;‘7 ‘,. : ;t..f.: f .:.2\.,‘i:;,: ; :_. proteins; -., In
&is
,pa@r.’
we’
&all.,
&&be
I om.. ~s~~~~~~~h~~~.,~~~.~.,,
preach. 7,s. to .evaluate stoichiometry, entige&.~~and .-kineti& ,&.&&t,edL~ with -.each of, tl&& h&e protein& and th&, -mktures:- : ....:‘, .:,: ,::;.;;r.y..L :.‘-....,:_,.;f$ :_ .. . . . ...: : ‘i-,‘.... t‘. II;;, ,.. ,’ ., :::. :,
..
both
a
... ..
,::; .7:-‘T.-+g :' .: ..'1 :_.,:._ .j:;,,.__'.~._j‘<, .‘:_.,, OTE“&d---a 'pt' d&i&de,,.'&O @&iT:eg
redox level. of the solution can. be coikhi~hilj;‘ ;,_A&$:.r&Uy,_ @@t&!l using mediator-titrarits, while simultaneous +tentio&etric and optical’-. measurements can be taken,
‘C
‘
392
Kuwana
and
Heineman
employed, for example diaminodurene, pyocyanine, anthraquinone sultetramethyl-+phenyl-enediamine (TlMPD), fonate, ferri-ferrocyanide, phenazine methosulfate or ethosulfate. substituted bipyridylium salts (com.monLy called viologens or paraquats), various indophenols and These mediators, in order to produce a stable, meaningful pO_ others. tential must exchange electrons with the b&component and also with the electrode. Thus, the total level of electron exchange must be sufficiently high so that a poised potential is achieved. A poised condition may not abvays be achieved (i.e. reports of potential drifting l*J3), which is indicative of slow electron exchange with the indicator electrode. The alternative explanation , sloW attainment of equilibrium in solution, is less IikeIy unless a corresponding drift in spectral features is reported. -
red
t
u Fig. r. Schematic diagram of eIectron transfer electrode (OTE or Pt) to or from heme proteins. For example, mediafor--titraElt A may be MV’+ or MV+.. mediator-titrant B may be ferricyanide-feocyanide, or O,-HIO. Heme protein A may be cytochrome c and B cytochrome c oxidase or others. The applied potential, U, governs the direction of the electron transfer.
OTE + Pt
Depending on the electrochemical characteristics and the value of the UO’, mediators also serve as cozclotnetric titrants in our studies. The two uses appear almost exclusive of one another. That is, for a mediator, the U”’ values between the mediator and the bio-component should be close together whereas, as a titrant, they should be far apart for completion of the desired reaction (equilibrium lies far to the right). The best situation is where one has a variety of mediator-titrants with UO’ values graded in steps of a IOO mV, or so, tbrough a range of for example -500 to +Soo mV. Then, two or more of these mediator-titrants can be utilized to esamine a particular b&component. From the change in optical absorbance of the b&component vs. the electrochemical change, accurate assessment of the equilibrium and hence of both n and UO’ of the b&component is possible, assuming the n value and UO’ of the mediators are accurately known. Unfortunately. our arsenal of electrochemically well characterized mediator-titrants is still limited. For
Energetics
and
Kinetics
of
Heme
Proteins
393
reductions, we have evaluated a series of viologens l4 which undergo a stepwise, one electron each (EE mechanism) electron transfer from the Eation to the radical monocation and then ,to the neutral species. The values for the two steps are sufficiently’ separated that -the radical can be .preferentially generated as the reductant. The UO’ .vab.res-range from -500 to -300 mV. For oxidations [Fe (CN)J3- has been, generated from [Fe (CN),]+ (UO’ = 424 mV). Viologens and the Fe-cyanides are nearly ideal mediator-titrants ;- it = I, good chemical stability, fast electron exchange with most components’and with the electrode, and low optical absorbance in most of the spectral regions where heme proteins absorb. TMPD (UO’ = 270 mV) has been widely ,used, although it does not have a simple two species equilibrium at pH 7. Molecular 0,, the physiological oxidant for cytochrome c oxidase, has been coulometrically generated at both the Pt and the SnO, electrode as a titrant in some of the studies reported here. Cytochrome c oxidase was isolated from beef heart mitochondria and purified according to C. R. HARTZELL. The heme a and Cu content were 14 pmol/g protein and 15 pmol/g protein, respectively. Horse heart cytochrome c was prepared according to MARGOLIASH.rs All reagents were high purity, biological grade. Spectra have been taken, usually at X-Y recording speeds, using an improved version of an earlier described Is dual beam, direct log absorbance readout, rapid scanning spectrophotometer [(Harrick Scientific Corp., Ossining (N.Y.)]. All potentials are corrected and reported, with respect to normal hydrogen electrode (N.H.E.).
StoicAiomed7y As a prerequisite to evaluation of the stoichiometry between bioelectron transfer components and to understanding the energetics and kinetics of reactions involving biocomponents, it is desirable to have an accurate assessment of the total number of electrons (12 value) transferable to individual components. Accurate and precise values for n .have been measured by the indirect coulometric titration method. Fig. 2 A shows the absorbance--charge (AA-AQ) plot for the reductive, followed by the oxidative, titration of cytochrome c odxidase. The methyl viologen radical cation, MV+ , was electrogenerated from MVZ+ (Uo’ =.~#6 mV) for the reduction and 02 was clectrogenerated at the Pt microelectrode .‘. for the oxidation. The fi value can-be calculated from the total charge or equi+lents required to titrate’ all of the oxidase or from the slope of the A:A-AQ plot according to equation I:
Kuwana and Heineman
394
where AE is the difference molar absorptivity (reduced - oxidized), in f&I-lcm- 1, b is the optical path length of the cell in cm, T’ is the. cell volume in Liter, and 91tis the slope in a.u./eq. Results for several heme proteins titrated coulometricahy are summarized in Table I. Since n values were known already for these heme proteins, the data principally serves to illustrate the precision and accuracy attainable by this coulometric method_ In some instances, the reductive oxidative titration was repeated through several cycles with very little variation in the resulting n value escept for cytochrome c oxidase which required about IO Ojo more charge during the initial reductive titration. The reason for this is presently not known.
Table
I.
CouIometrically
determined
n values
72
Component
Value -
I ( kidation
Reduction
a. Reductions (C%J3--* phosphate 6. 30.6 t.
52-7
d. 16-7 avg.
I.OZfI 0.04 I.19 r_I4+0.08 2_15+0.3 j
by electrogenerated except oxidase by 0,. buffer at pH 7.0.
~_LLM myoglobin, p&f modified to 26.7 @f 6 solutions.
e. 5.5 PM cytochrome
0.5
M
MV+; All
Ka[Fe(CN)
cytochrome cytochrome c oxidase,
0.03 I-04 1.03 & 0.09 2.04+ 0. ro
o_gg&
t, 0.5 c, 0.5 0.6 mM
fi.
7
‘,*
Icl Xeduction -_
.-
Myoglobin b Modified cytochrome GC Cytochrome cd Cytochrome c oxidase 6
heme.proteins
of some
r.oo+ 0.01 o-97 r.oz~o.14 r.g8+_0.21
2 3xidation
2
I.OZ+-0.01 0.95
r.o4+o_os 2..14+ 0.24
oxidations by electrogenerated [Fe solutions contain 0.1 M N&l, 0.1 M J, avg.
m&f mM
3 solutions.
MVzf, MVzf,
0.5 m&f [K,Fe(CN) 0.5
to 2 mM
J_
KdFe(CK)J,
MV”-+-, 0.1 O/e Tween 20, avg. 4 or more
solutions.
The n values measured by indirect. coulometric titration represent the total number of electron-equivalents consumed by the heme protein per mole of the optically monitoied species. In contrast, the it value measured by potentiometric titration is specific for the specks whose spectral band is used for calculation of the ratio of reduced to oxidized forms. Consequently, n values which are obtained by the coulometric method may differ from those obtained by the potentiometric method. In the. case of cytochrome c oxidase, a value of 2 e- per heme a (one Cu per heme is titrated simultaneously) is obtained by the’ spectroelec-
Energetics and Kinetics of.,Heme Proteins
39.5
trochemicai coulometry whereas a I e- value is obtained by the potentiometric method I7 since it is only the heme n constituent whose ratio of redox forms is being monitored at 605 nm.’ The indirect coulometric titration has several advantages over conventional chemical titration procedures for measuring n values. These are: I. solution conditions remain essentially invariant during the titration ; 2. smaLl increments of the titrant (charge) can be added accurately and conveniently at any repetitive rate ; _?. the bio-component can be repetitively cycled through various oxidake or reductive levek rapidly (few minutes, if necessary). The supply of titrant is infinite since its original redox state is regenerated by electron transfer with the b&component. athough the electrical potential applied to the electrode is thermodynamically sufficient to cause direct electron-. transfer to or from the bio-components, the overpotential for transfer is such that the electron is transferred for all practical purposes quantitatively through the mediator-titrant. -.
Energetics
.-
The potentiometric method has been employed extensively to determine UO’ values of both puri@ed and intact components of the oxiThe concentrations of the components dative phosphorylation chain. have been assessed spectrophotometrically during titration and n and UO! values, ‘evaluated from. the familiar nernstian plots of Urn-~ ,vs. the appropriate logarithm of concentration ratio (expressed in optical absorban’ce) . UO’. values of heme proteins can also be measured by evaluating solution equilibria between the b&components and another redox component- whose UO’ ,is accurately : known by independent measurement. Assuming a quantitative equilibrium transfer of charge from :the titrant to the bio-component, it is possible to evaluate the energetics (UO’) of a bio-component from analysis of the AA-AQ curve cbtained from the The applicability of this method was indirect coulometric method. demonstrated by ,HAWKRIDGE 7 for cytochrome c, where the mediatortitrant system of methyl viologen (UO’ =‘-446 mV ZIS. N.H.E.) and The, UO’ ferri-ferrocyanide ( UO’ = 424 mV OS. N.H.E.) was employed. f6r cytochrome c was evaluated as 258 & 17 mV ZLS.N.H.E. In ordTr to obtain accurate values by this method, the UO’ of the reference niediator (ferri-ferrocyanide in this case) sliould be within ca. 200 mV of the bio-component’s UO’. The case of cytochrome -c’ oxidase is complicated by the presence’ of four redox centers per molecule, two hemes and, two Cu. Information concerning the redox potentials of these four components of cytochrome c can be inferred from the experimental AA-AQ curve shown in Fig. z A. In this titration the UO’ .of the two titrants are sufficiently removed from. those ,of the bio-componentS that their influence. on the AA-AQ
Kuwana and Heineman
396
Fig. 2. o-6 m&f MV+. A. hAmor AQ plot of coulometric titration of s-5 g&f cytoclqome G oxid-. 0.1 M NaCl. and 0.1 O/O Tween 20 in 0.3 M phosphate buffer pH 7-o. No correction for IT+ sidual charge. (0) Reduction by PUN+: (0) Otidationby 0,.
&l(05-AQ plots for cytochrome G oxidase. (-) UaraL - UO’cUL or U”‘_+= UO*cUII = U”‘ar= U”‘cU,; (- -) Cl”‘+ =
B. Computer simuIated ua*cLla
-
UO’CU
L
--
280
; (--+-)
Ua'+
=
350,
ZYa~
=
U”‘Q~
=
210,
UJ’aH = U”‘CU~~. 350,
U”‘Q~
U”‘aL
=
=
2.10.
310.
curve is negligible. Only two situations will give a straight-line plot of AA-AQ as observed in Fig. 2 A : I. the standard redox potentials of all four components are ider+cal; 2. the heme potentiafs are separated with each of the Cu potentials separated identically with the hemes. The AA-A0 curves for this multiredox heme protein can be computer simulated “using the four Nemst equations : U =
P’heme uH-
59
log([heme
a,,(red)] /meme
u =
U”‘heme aL -
59
log(@eme
+(red)] /[heme aL(ow
(3)
u =
Uo’cy,
I[CuH(ox)31
(4)
-
u = SO’c uL-
59 logKCu&ed)l
59 log~CCu,(red)]/CCu,(ox)])
4I(om
(4
(5)
As the redo-u potential, U, reflecting the redox state of the solution, is var-
ied from a value in which all the components are in their oxidized form to a value in which they are in their reduced form, the resulting ratios of each
component (each heme and Cu) can be computed in terms of the consumed charge and of the change in the optical absorbance at 605 nm. Results
of such simulations are shown in Fig. z B where UO’ values of 350 and
Energetics
and
Kinetics
of Heme
Proteins
397
210 mV were assigned to the two hemes and the values of the two copper potentials were varied as indicated in the legend. It is estimated that a difference Of a u”‘Cu from a u”‘h eme a by IO mV, or more, should be experimentally detectable by the deviation from linearity of the AA-AQ curve. Deviations from linearity of the type noted in Fig. 2 B have not been observed (i.e. plots reported by KUWANA 8 and VAN GELDER lS are linear in the intermediate region of titration). It should be noted that the reported values of the Uo’cu based on the behavior of the S30 nm band would definitely produce deviation from linearity in these plots. In Fig. 3 the AA-AQ is plotted for the indirect coulometric titration of cytochrome G oxidase in the presence of MV2+ and ferri-ferrocyanide. lg In this case Uo’ values for the redox components of cytochrome c o-xidase can be determined from the deviation from linearity caused by equilibrium with the ferri-ferrocyanide couple. The solid line is for a computer simulation of the AA-AQ using the UO’ values of 434, 350, 350, zzo, and 220 mV for ferri-ferrocyanide, heme aH, Cu,, heme aL, and Cu,, respectively. The calculation is corrected for the weak chargetransfer complex 2O formed between [Fe (CN),]+ and MV2+ dication. The effect of varying the Uo’ value of Cu, is illustrated in the same figure. The agreement between the calculated and experimental results is excellent for the values of UO’ being 350 and zzo mV for the two heme a-Cu pairs. The same experiment was repeated with TMPD replacing ferri-ferrocyanide as a mediator. Is The results appear to be identical within experimental error.
0.20 -
0
h
-s 0.16
is ”
-
: 0.12
-
0.08
-
o_oc
-
0.00
-,
g 9
.i ” 1 00
I 40
0
\
Qneq/cm3
I 120
I 200
Ii0
Fig. 3. AA-AQ plot for reductive MYa+, 0.5 mM [Fe(CN)d]s-,
coulometric titration of 9.3 FM cytochrome c oxidase. 0.5 m&I and 0.1 o/o Tween 20 in 0.3 M phosphate buffer pH 7.0. Corrected
for any background charge.
A. Computer simulated for UO’+ =
U”‘C~, =
(0) experimental points. B. Computer simulated for U”‘+
350, UO’cCH=
=
350 mV, 280.
U”IaL= U"*+
=
UO’cUL= U”‘cUL =
220
220
mV. mV.
Kuwans
39s
0
2
4
and
Heineman
6
Titration of 17-5 @Z cytochrome c and 6.3 PM cytochrome c oxidase by reduction with MY+-. Solution conditions same as in Fig. Z. [Upper left hand comer insert : AA-AQ plotsfollowing Main figure : y 605 and 550 nm absorbance bands ; charge corrected for background]. 0 experimental points. Solid .=IA45501(r~~ % -AA,,,)]. x = kL4,,/(100 % --A&,,)]. lines simulated for CT”‘+, 1: 350 mV, U”‘+ = zoo. 210, and 220 mV. UO’w~, c=250
mV.
_
Uo’ values of the two heme a components of cytochrome c oAridLe have also been determined from titrations of mixtdtes of cytochrome c and cytochrome c o_xidase. The AA-AQ curve (monitored at 605 and 550 nm) for such a titration is shown in Fig. 4 (insert). It can be concluded from the shape of the absorbance change at 605 nm that the ZP’ of cytochrome c is intermediate of the UO”S of the two heme a’s. The
Energetics and
of ,Henih
.Kiiieiiics
Proteins
399
_
behavior of the-.ofitically monitoredhcmes can -be separated from. theCu% (which also consume charge) by .pl&ing I:the.: logarithms:.of : the: titios' :ofa the reduced to. oxidized -forms as sho&n~,incFig.. 4; :-The-d&a are plotted as., log[c(red)/c(ox)] vs;.:‘,log[~~,(red(/a&(ox)] : i w h ere : ‘+&)~c(ox)~.-+ = ‘AA&/ g&(red) /I% .&, AA dljs /(qjo.-o/d~~ (IO0 70 --AA_& .. -and -AA aas). AA representing the percentage of the total absorbance changer The ( &A.lting sigmoid-shaped cur& : is characteristic :‘of ,::-a: ~‘uiultiredox system_. .,’ I :. _. ., : .. ...,, 1 ., _ : ,..:‘..:~I\‘:. .:.:,-;-!..._:;_:c:i,_i ” Similar. curves can be obtained: by calculating -the log terrhsof .the .. fohowing Nernst -equations,- as the potential .U of- theasystem’: (solution) 2; ;, ! is varied : :
u =
UQ’cgt
j.
u=
UQLne~
u
=
c
59 log[c(red) /c(cox)]- -.J’
-
-
aL -
cJfJr,,
59
59
Jogpeme
.
(6)
zz,(red) /heme a,(ox)]
Iog@eme &(=e&/heme
(4
%(0x)]
(3) ,
where the -subscripts H -and L ‘&fei- to high. -and .loii;. potential :hem&., Log[b(red) /c(o)r)] ‘is then_ plotted against log([&(red)- $L &@l)] /[aL(o,x)“q. assigning +a++ -t %&m)--me solid. Curvg- in Fig. 4 were obtained':by of, W&r = 250 mV, vT’hcmc+ = 35o ‘mV’: and’ varying -the.;.value of that’ ‘th,e cu.+ :are @te U?.‘hall= 5. from zoo ‘to 220 mV_ : _Jt is ,ap&+nt sensitive. to- changes in U”‘. The .U?’ valuesof ra, and e&re &z&ned_ 35o and ZIO .mV; respectively;: based
‘.
m+-2 . MV+-
+
+_,,e~.&-_~+*
.‘I
cytochrome ca
kf,-
*
kb ':
.
.__I. I.
MV+*..+
-‘. i
‘.- -._ I.‘. ,,,__1: i.r-,iy]’ : 1.
cytochrome c&
.:.:. ,-(8)
_._: .: -....! i .: ;,I ;, >,c ;. _:;.:-:,;_:'L_,..: by,the.,~m~
\t&' c&Z the electrogenerated ‘kV+ : is '~&&d.&& dizzed cytochrome c. The rate constant,. kf, for the homogen&%rs.- e:
where
Kuwana
400
and
Heineman
transfer can be conveniently evaluated spectroelectrochemically by optically monitoring at set wavelength for either NV+or reduced cytochrome c as a function of time during a chronoamperometric experiment. Spectrochemical evaluation of rate constants for such mechanisms was demonstrated earlier and extended to an enzymatic electron transfer by ITO. 2z A discussion of the concentration-distance-time (c-x-t) and absorbance-rate constant-time (A-k-t) profiles which were computer calculated by the method of digital simulation, 23 will be. presented for the purpose of cIarifying the spectroelectrochemical approach. In a generalized EC catalytic reaction scheme,
where A* is electrogenerated at a diffusion controlled rate, the c-x profiles as a function of time, t, are shown in Fig. 5_ The initial conditions of the esperiment can be tied : for this simulation, at t = o, the concentration of A was set equal to 2. The concentration of A* depends on the diffusion coefficient of A, the concentration of 2 and the value of kf, assuming kf+ kb. Explicit in the mechanism is the high overpotential for the heterogeneous electron transfer to 2 so that it reacts at the electrode at a negligible rate compared to A. This condition ap-
-c e-
A-A* Li__________*
A-+2
-K-A
+
27:
k =lO’ 1.0
-----A2
P
E
Y
r”
0
ms
s
0 Distance,
cm
Fig- 5_ Concenbation+liscance profiles for species, A, Af * Z and 2’ as a function of t -0, I ms, 4 ms and S ms for second order EC catalytic mechanism. Rate constant k = IO' ; at t I o, [A ] =
[Z] =
x0-’
M_
Energetics-and Kinetics of I Heme Proteins +=+A%--,
_-.._-. . = ?OC
&=O,Y.~
“.~__-“_‘__“
A-+Z&i\+Zz
_ .rr-6
401.
:
,Af.
.;y
.. . .
:‘.
‘.,..
ms 0
1
2
4
3
‘_
..
._ Fig. 6. Absorbance as a function of time cokpnted for, varioi v&es of ,k for second ord& Ed: &atalytic mechanism during potential step (chronoamperom&ic) expbiment. !$ecie& k* : a 1.04. At L= O, [A] = [Z] = x0-8 M ; DA = 101 cm* s-1. CA* 0pticaUy monitored. ..
to be fulfilled f or many of the nalxked hem& proteins; The ~o&cal absorbance, A, changes during the electrolykis. ,tjrqe. depending on kf atid plots of A - t for various values of kf are’ shown in Fig; .6.., Fck ltige values of kf and/or small values of molar’absorptivity of the &onitored species, it may be necessary to signal average’ the-optic+ respok during’ repetitive electrochemical pulses: or utilize internal reflection techni@s.
pears
Fig. 7.
_. .. *; *_
:
b+
..
‘.
Change in opticalabsorkance~+3 a fm$ion of time during cqu~pav~i .p&+ti$ pulse. A. Monitoring at 630 nmthe for7 mation and removal of &IV+-: B: In the presence of cytcuzhrome c. Absarhance is signala==@ (135op*) =+outfmm Puke Iength 8 PAR Wav.efo+ Ednctor.
-
Time -_)
ms, io&
sqnareywav~ &,&tion’of,d$
630
nm
Dm+z
repeated_ every &_--&fi’&$&&
=
D~c-r.r~10*~?s-‘(ref.25)r
6.86 & xd4
‘2 s_fot‘
&in- ‘ii+’
403
Knwana
and
Heineman
Preliminary results 24 of the A ,- t patterns for the reaction of &IV+* with oLxidized cytochrome c are shown in Fig. 7. Curve A is the signal averaged optical response for monitoring &IV+- during a 8 ms square wave electrochemical pulse which was repeated every z seconds for a total duration of 45 minutes (1350 pulses). The signal was averaged using a PAR \V.AVEFORM EDUCTOR (100 channels, maximum time resohrtion APPLIED RESEARCH, Princeton, N-J.). I ps per channel ; PRIXETOX In curve B of the s+me figure, the experiment is repeated with equal moIar concentration of cytochrome c~ present. The rising portion of the absorbance (8 ms duration) corresponds to when the U,,U~ is a value such that MV** is being produced at a diffusion controlled rate ; it reacts with the oxidized c as it moves toward the bulk solution phase. When Uapp~d is stepped back to a value such- that reaction (7) is reversed, the absorbance decreases with time. The Kf of reaction (S) can be analyzed from both the forvard step time (8 ms), or from the decreasing portion of the absorbance. Only the former will be discussed here. The normalized absorbance (A -=B/A-~A) is computer-calculated for various values of kf and t, using the initial concentrations of &IV*: and cytochrome cO~~ and the known diffusion coefficients of the tivo species. By fitting the experimentahy determined normalized absorbance to the computer-simulated normalized absorbance, ‘the value of kf Ca.c be’obtained. For the conditions of the esperiment (?ee Fig. 7). the rate constant was evaluated as equal to 5 (-& z) x .IO"I moF s+. This value is an order of magnitude larger than the rate constant for the cytochrome cdUc~ - cytochrome c,a self-exchange and an order of magnitude less than the rate of [Fe (CN),] 3--o_xidation of cytochrome ctdUd. Because the driving force of reaction (S) is large (AGO’ = -16 kcal mol-r as estimated from the UO’ values of the two redox couples) the rate is espected to approach diffusional rates except for any steric factor involving the reactants. SUTIN, 28 assuming transfer through the exposed edge of the prophin ring, estimated that this electron transfer site occupied about 3 o/0of the total surface area of cytochrome c. Assuming a collisional model and a 3 o/0steric factor, the estimated rate is on the order of 108 Z mol-1 s-l_ Obviously, the measured rate is considerably less than this. To quote SUTIN.~~ “ ...mechanism of electron-transfer to and from cytochrome c is a challenging problem and further studies using a wide variety of oJxidizing and reducing agents are necessary before any mechanistic assignment can be made with confidence “. Further spectroelectrochemical measurements of kinetic parameters involving mediator-titrants with cytochrome c and cytochrome c ,oxidti& are in progress. Discussion
For the purifieP cytochrome c oxidase, there has been considerable discussion regarding the similarity or dissimilarity between the two hemes. Also, there has been suggestion that cytochrome c oxidase titrates
Energetics and Kinetics of Heme Rote&
403
as one component (all UO’ values identical) in the absence of cytochrome c and that the presence of c or ferricyanide-ferrocyanide splits the Uo’ values of the two hemes. We would like to make .the following statements with regard to these two points : I. In a reductive potentiometric titration, both hemes will titrate as one component when a strong reductant is used. If a mediator with UO’ more negative by IOO mV or more (e.g. phenazine methoslilfate) is used, the hemes will still titrate as ooze optical component. The reason for this is that the two hemes are not well differentiated optically. Only if one or both Cu titrate at a potential sufficiently apart from the heme potentials, will there be a curvature in the AA-AQ plots (e.g. Fig. z B). 2. Because the two hemes titrate at different potentials in the presence of cytochrome c, or other mediators, doe-s not necessarily mean that these two hemes cannot be identical in the fully oxidized or fully reduced states. A possible reason for UO’ differences observed experimentally is that each electron transferred alters the charge and the electron density distribution, which consequently affects the GO’ for the next electron transferred. As suggested by several workers, 27*2athere appears to be evidence for heme-heme interaction. If so, it is expected that the potentials for the half-reaction involving the second heme titrated will be different from the UO’ of the half-reaction of the f&t heme titrated, even if the hemes were initially identical in the fully reduced or oxidized states. Interpretation as to the heme-heme interactions from potential data is tenuous, however, since equilibration between the hemes may occur through electron exchange with a mediator, or mediators (in case of potentiometric titrations), and not through direct electron exchange between the hemes. There is little information available on the latter. The stoichiometry and energetics correlated to tbe available kinetic data strongly suggests that the addition or removal of electrons occurs stepwise in two I.1 electron pairs. Each pair of electrons corresponds to a heme-Cu unit. From kinetic studies, 29 it appears that two electrons are added to the fully oxidized oxidase initially at a rapid rate. Thus, the rates of transfer to and from oxidase for each. pair (not to imply simultaneity of the two electrons transferred) may be different. If equilibrium titrations are performed on the time scale of several minutes to hours, what may be observed are the electrons filling or emptying cytochrome c oxidase according to the thermodynamics of the,components with respect to each other. However, if transfers are made on a much shorter time scale, kinetic differentiations may appear with respect to the site, or sites, of electron transfer. The problem is further complicated by the possible dependence of the mechanism of transfer on the type of reactant. Continued studies are certainly warranted and needed. The assignment of Uo’ value to the hemes and Cu’s is summarized in Table 2. Selected literature values are included for comparison purposes. It should be noted that the heme value of ca. 280 mV reported for the situation where the hemes are supposedly identical is an average of the Uo’ values of heme a= and heme q. Similarly, the potential for
Comparison
cyt a,
cyt a
2W.5) 225 0.0) I
as single component
designated
b. Frequently
c, Titrated
1.0)
225 (N I) 232 (- I) 55 W)
designated
ao,a1
la
35o&5 (1.0) 3503- IO( I .o)
high b ________-__
c 375 (1.0)
.364 (N I) 347 (N I) 251 (N I)
335-36oW)
280(x 80) c
(LO) 30( LO)
zoo-280(
2202
zrok5
10~ a
____
heme a
30(1.0)
3503- IO(I,O)
c oxidase.
Conditions
--.--_
highly purified partially purified
5 x 10-a M [Fe(CN) J85 x 10-4 to 10-a M [Pe(CN)JOo to 10-6 M fFa(CN) J*-
cyt c absent. cyt c present
cyt c present [l?e(CN)Js’ present
-------
e. Based on 830 nm band and EPR
d. Based on 830 nm band
220&
low
of U0’ values for purified cytochromc
a. Frequently
WILSON
CUSANOVICH
VAN GELDER l*
This paper
Source
Table z,
Energetics and
Kinetics
of Heme
Proteins
405
Cu determined from monitoring the 830 nm band appears to be near the average of the UO’ for Cu, and Cu,. Preliminary spectroelectrochemical coulometry at the 830 nm band shows close parallelism to the 605 nm band of the hemes.
AcknowIedgements
This investigation was supported by PHS Research Grant GM 19x81 and initiated during tenure of T. K. (NIH Special Research Fellowship I F03 GM 48486) at the Institute for Enzyme Research at the University of Wisconsin, Madison, under Professor H. BEINERT. W. R. H. acknowledges support from RESEARCH CORPORATION,COTTREL Grant 6720. The authors satefully acknowledge discussions with C. R. HARTZELL. Cytochrome c and cytocbrome c oxidase were provided by C. R. H. Data provided by L. MACKEY and M. FUJIHARA are acknowledged. The authors appreciate preprint copy of paper by CUSANOVICH and WHARTON.
1 2 3 4 6
6
7 8
8
10 11 12 18 14
A. LEHNINGER.
BiochenzisCry,
Worth
Publishers
M.R. LEMBERG. Physiol. Rev. 49, 48 (1969) T.E. KING, H.S. MASON and M. MORRISON, Systems, Wiley, New York (1965)
(1970)
Ed. Oxiduse and Related Redox
K. OKUNIJKI, M.D. KAMEX abd I. SEKUZU. Eds. Sfrucfure and Function of Cyfochromes, University Park Press, Baltimore (x968) E.C. SLATER, B.F. VAN GELDER and K. MINNAERT, in Oxidase and ReZafed Rsdox Systems, T.E. KING, H.S. MASON and M. MORRISON, Eds., Wiley. . New York (1965) T. TAXANO, R_ SWANSON, 0-B. KALLAI and R.E. DICKERSON, in Cold Spring Nor&or Symposia on Quantifaotiue biology, vol. 36. Cold Spring Harbor Lab-
oratory (1972) F-M_ HAWKRIDGE
and T. KUWANA, Anal. Chem., 45, IOZI (1973) W.R. HEINEMAN, T. KUWANA and CR. HARTZELL, Biochem. Biopliys. Res. Commun. 49, I (1972) ; ibid. 50, 892 (rg73) N. WINOGRAD and T. KUWANA, in EZecfraanaZyticaZ Chemistry, A Bard, Ed., Marcel Dekker, New York, in press vol. 7 J_W. STRO JEK and T; KUWANA, J. EZectoanaZ. Chem. InterfaciaZ JTkctrochsm. 15, 471
(1968)
A.H. C&WELL and B.C. PRESSMAN, Arch. Biochem. Siophys. 125, 318 (1968) M.A. CUSANOVICH and D.C. WHARTON private communication M. ERECINSXA. D.F. WILSON. Y. MUKAI and 33. CHANCE, Biochem. Res. Commun. 41, 386 (1970) E. STECKHAN and T. KUWANA, unpublished results
Siophys.
Kuwana
405 15 16 I7 18
19 20 21 22
23
24 25 26 27
28 29
31
Heineman
E. MARGOLI~~H and A. SCHEJTER. Ada. Profei= Chem. 21, 114 (1966) J.W. STROJEIC, G. GRUVER and T. KUWANA, AnaZ. Chem. 41, 4Sr (1969) P.L. DU~ON, D-F. WILSON and C_ LEE. Biochemistry 9, 5077 (1970) A-0. MUIJSERS, R.H. TIESJEMA, R.W. HENDERSON and B.F. VXN GELDER, Biochim. Biophys. Acfa 267, 2x6 (1972) L.N. K~CKEY, T. KIJW_~X_~ and C-R. HXRTZELL, FEBS Letf. in press A. NAKAHARA and J-H. W-G. J_ Phys. Chem. 67, 496 (1963) K. MINNAERT, Biochim. Biophys. Acla 110, 42 (1965) M_ ITO and T. KTJWANA, J. EZecfroanaZ. Chem. Infevfacial Elecfrochem. 32, 415 (1971) SW. FELDBERG,
in EZecfroanaZyylicaZ Chemisfry, vol. 3. -4-J.Bard, ed.,LMarceI Dekker, N.Y. (1969) Data provided by M. FUJIEARA, Ohio State University A. EHRENBERG, Acta Chem. &and. 11, 1237 (1937) N. SUTLN, Chem. Brif. 8, 148 (1972) D.F. WILSON,. J.G. LINDSAY and E.S. BROCKLEHURST. Biochim. Biofihys. Acta 256, 277 (1972) D.F. WILSON and J.S. LEIGH, Jr., Arch. Biochem. Biophys. 150, 154 (1972) Q.H. GIBSON, C. GREENWOOD. D-C. WHARTON and G. PALMER, J. BioZ. Chem.
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D.F. \VILSON, P.L. DunOI<, M. ERECINSKA, J.G. LINDS_%Y and N. SATO, Accounts Chem. Res. 5, 234 (1972) T. Tsrrirw~r and D.F. WILSON, drch_ Biochem. Biophys. 145, 149 (1971)