144

Biochimica et Biophysica Acta, 1140 (1992) 144-156 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2728/92/$05.00

BBABIO 43727

Reduction kinetics of the four hemes of cytochrome c 3 from Desulfovibrio vulgaris by flash photolysis Hideo Akutsu 1, Jo H. Hazzard, Robert G. Bartsch and Michael A. Cusanovich Department of Biochemistry, Unicersity of Arizona, Tucson, AZ (USA) (Received 8 May 1992)

Key words: Laser flash photolysis; Cytochrome c3; Phototitration; Reduction potential

The reduction of the tetraheme cytochrome c 3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenborough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heine and the reductant can be described by outer sphere electron transfer theory. Thus, the very low reduction potentials of the cytochrome c 3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c 3 arc consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.

Introduction The cytochromes c 3 form the Class 3 cytochromes which differ from the more commonly studied Class 1 and Class 2 cytochromes (such as mitochrondrial cytochromes c and prokaryotic cytochromes c', respectively) by a number of unusual properties that make them valuable experimental materials for understanding biological electron transfer. These cytochromes are found most commonly in sulfate-reducing bacteria of

i Present address: Department of Physical Chemistry, Faculty of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240, Japan. Abbreviations: cyt c3, ferric species of cytochrome c 3 unless specifically described as reduced cytochrome c3; 5-DRF and 5-DRFH', oxidized and one-electron-reduced 5-deazariboflavin; PDQ 2+ and PDQ +', oxidized and one-electron-reduced propylene diquat; MV z÷ and MV +', oxidized and one-electron-reduced methyl viologen; NHE, normal hydrogen electrode. Correspondence to (present address): J. H. Hazzard, Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis MO 63110, USA.

the genus Desulfovibrio [1,2] and are believed to function as electron carriers for hydrogenase [3,4]. To date, the cytochromes c 3 from four species have been studied in terms of both structural and physicochemical properties: D. vulgaris Miyazaki F and the highly (86%) homologous D. culgaris Hildenborough, and the more diverse D. desulfuricans Norway and D. gigas Gigas. Perhaps the two most important distinguishing characteristics of Class 3 cytochromes are: (1) the presence of multiple heine groups which have (2) very low reduction potentials (reviewed in Ref. 5). Interestingly, solid films of reduced cyt c 3 exhibit a very high conductance [6,7], suggesting electrical properties which might have useful applications in bioelectronics. It is of special interest to study the interaction between the hemes in the Class 3 cytochromes in an effort to define mechanisms for intramolecular electron transfer. In the Miyazaki F, Hildenborough, and Gigas cytochromes there is considerable h e m e - h e m e interaction which permits rapid intramolecular exchange of electrons (exceeding the N M R timescale or (1.5-7.8). l0 s s-1 for Miyazaki F as referenced in Ref. 8), while the intermolecular electron exchange rate is slower than the N M R timescale [9-11] (1 • 104 M - t s - t for Miyazaki F

145 [8]). For the Norway cytochrome, however, the intermolecular exchange rate is faster than the NMR timescale, and there is no indication of rapid intramolecular electron exchange [12]. Substantial changes in heme reduction potentials (up to 50 mV) upon partial reduction of the four hemes have been observed and quantitated for the Miyazaki F cyt c 3 [9]. From the reported crystal structures for the oxidized Miyazaki F and Norway c 3 cytochromes (Refs. 13, 14, respectively), it is clear that the orientations of the four hemes are almost identical for the two proteins in spite of their low sequence homology. Still, the Norway cyt c 3 appears to differ markedly in its properties from the Miyazaki F and Hildenborough cytochromes. The macroscopic reduction potentials of the Miyazaki F cyt c3, which relate to the five macroscopic oxidation states of the molecule, have been measured as -240, -297, -315 and -357 mV vs. NHE [15] and the microscopic reduction potentials of the individual hemes have been estimated by 1H-NMR measurements in which hydrogenase was used as the reducing agent [9]. The 1H-NMR data indicate that the environment of each heine group is altered as the surrounding hemes are reduced [9,16]. For the Miyazaki F cyt c 3, reduction potentials have been assigned to all four hemes by the use of 1H-NMR: Heine IV has the highest reduction potential, followed by Heme I, Heme III, and finally Heme II with the lowest reduction potential [9,17] (heme numbering for both the Miyazaki F and Norway cytochromes according to amino acid sequence [18]). For the Norway cyt c3, reduction potential assignments have been made by single-crystal EPR measurements: Heme III has the highest reduction potential, followed by Heine IV, Heme I, and Heme II with the lowest reduction potential [19]. For the Miyazaki F cyt c3, the reduction potential decreases with increasing exposure of the heme to solvent, as anticipated in previous discussions [20,21]. However, for the Norway cyt c 3 the correlation between solvent exposure and reduction potential is not as consistent, suggesting that additional factors are substantially influencing the reduction potentials in this cytochrome. Indeed, a number of factors may play a role in controlling oxidation-reduction potentials in cytochromes: axial ligation of the heme, local dielectric, electrostatics, ligand orientation and hydrogen bonding [5]. In addition to reduction potential determinants, steric, electrostatic and hydrophobic factors will define the interaction domain on the surface of the cytochrome for external reactants [5]. An accurate assessment of the quantitative contribution of each of these factors to the overall process of electron transfer remains a long-term goal at this point. However, within the past decade, rate constants for electron transfer in a wide variety of biological molecules have been mea-

sured and their correlation with the reduction potentials of the respective proteins has been demonstrated (reviewed in Refs. 5 and 22). The correlations are based upon the Marcus theory of outer-sphere electron transfer [23,24] and, in some cases, electron tunneling [25] (see Ref. 22 for more extensive discussion), which were established from data obtained by measuring the kinetics of electron transfer from the semiquinones of free flavins (and flavodoxin) to several series of homologous c-type cytochromes, HiPIP's (high-reductionpotential 4Fe-4S ferredoxins) and blue copper proteins. From these measurements, the contributions of both electrostatic and steric effects to the electron transfer rate constant have been demonstrated within a given series of homologous proteins. Because of their unique redox properties, it is interesting to extend these observations to the Class 3 cytochromes, and in this report the reactions of Miyazaki F and Hildenborough cytochrome c 3 with the 5-deazariboflavin semiquinone (5-DRFH, E m = - 6 5 0 mY), reduced methylviologen (MV ÷', E m = -446 mV) and reduced propylene diquat (PDQ ÷, E m = -550 mV) are measured by laser flash photolysis and compared with similar data for other c-type cytochromes. These reducing agents act as effective reductants of the low potential cytochromes c 3 while providing different chemical and electrostatic properties which can be used to probe the interaction domains of the individual hemes. The results of this study for both the Miyazaki F and the Hildenborough cytochromes c 3 are compared to the heme reduction potential assignments previously made for the Miyazaki F cyt c 3 by NMR measurements. In addition, the presence of an unusual ionic strength dependence of the rate of electron transfer is demonstrated in these Class 3 cytochromes. Preliminary results of this work have been published in abstract form [26]. Materials and Methods

Cyt c 3 was purified from Desulfovibrio vulgaris Hildenborough and Miyazaki F strains. The former was purified as follows. Pooled basic cytochrome fractions were passed through a Sephadex G-25 column equilibrated with 1 mM potassium phosphate buffer (pH 7.0). The proteins were then chromatographed on a CM-52 cellulose column, using a linear gradient, 10 to 100 mM potassium phosphate buffer (pH 7.0). The cyt c 3 fraction which eluted between 40 and 80 mM potassium phosphate (pH 7.0) was concentrated by pressure dialysis using an Amicon YM-5 membrane. This fraction was then chromatographed on a Sephadex G-50F column equilibrated with 100 mM NaC1 in 20 mM Tris-HC1 (pH 7.3). The best fractions of cyt c 3 were then chromatographed on a hydroxyapatite column using a linear gradient from 10 to 100 mM potas-

146 sium phosphate ( p H 7.0) in 100 mM NaCI. Cyt c 3 eluted between 80 and 90 m M potassium phosphate (pH 7.0). The cyt c 3 fraction was concentrated and desalted using an Amicon YM-5 filter. The purification of Miyazaki F cyt c 3 is given elsewhere [16]. The purity index (A552[red]/Az8o[OX]) of both proteins was 3.0. The extinction coefficient of reduced cyt c 3 at 552 nm was taken as 29 raM-~ c m - ~ / h e m e , which was determined for the Hildenborough cytochrome by the alkaline-pyridine-ferrohemochrome method. T h e same value was obtained by spectroelectrochemical titration (Niki, K., personal communication). 5 - D R F and P D Q 2+ dibromide were provided by Prof. G. Tollin. MV z+ and deuterium oxide (98%) were purchased from Sigma Chemical Company. Absorption spectra were obtained with a Cary 15 spectrophotometer modified for computer control by On-line Instrument Systems (Jefferson, GA). Laser flash photolysis was carried out at room temperature using an N2-dye laser (Laser Photonics) emitting at 386 nm (BBQ dye, Laser Photonics), which excites 5 - D R F to produce its semiquinone species 5D R F H in the presence of excess E D T A [22]. 5 - D R F H then serves as a reductant of cyt c 3. Thus, all experiments reported here were carried out in the presence of 5 - D R F (60-120 ~ M ) and 10 m M E D T A (or semicarbazide). The 5 - D R F H ' species will also reduce either MV 2+ or P D Q 2+ within 1 Izs, which in turn can reduce cyt c a. Electron transfer from MV + or P D Q +" to cyt c 3 outcompetes the direct reduction of cyt c 3 by 5 - D R F H in these experiments as reduction of MV 2+ or P D Q 2+ by 5 - D R F H " is diffusion-controlled and the concentration of MV 2+ or P D Q 2+ was 2 mM, in large excess over 5 - D R F H ( < 0.7/~M generated per flash), as previously described (28-30). All kinetic experiments were performed under pseudo first-order conditions with the concentration of oxidized cyt c~ heine 2 - 5 / ~ M . The buffer solution in a rubber septum-sealed cuvette was bubbled with oxygen-depleted and watersaturated argon gas for an hour to remove the residual oxygen. The anaerobic condition established by this procedure was checked by monitoring the stability of the partially reduced state. The reduction of cyt c 3 by 5 - D R F H and P D Q +" was monitored at 569 nm where the major contribution to the absorbance change is from the cytochrome heme. In the presence of methyl viologen, the reaction was monitored at 600 nm, where MV ÷ shows strong absorption and the contribution of the protein absorbance is negligible. It was confirmed by changing the monitoring wavelength that the kinetics of MV + oxidation are the same as that for cyt c 3 reduction. Partial reduction of cyt c 3 prior to laser flash photolysis was p e r f o r m e d by illuminating the sample (containing cyt c 3 and the reductant(s)) at a distance of about 12 cm with a 35 W tungsten lamp for 1-30 s

intervals prior to flash photolysis. The concentration of remaining oxidized heme was determined spectrophotometrically before each flash photolysis experiment, and was checked immediately following flash photolysis to ensure that the concentration of oxidized heme had not decreased. Phototitration experiments were conducted with 5 - D R F H " and P D Q +' only, the latter being used instead of MV + because of the greater potential difference between P D Q +" and the lowest reduction potential heme of cyt c 3. The use of P D Q +" was therefore expected to minimize any reverse reactions between the electron donor and the lowest reduction potential heme of the cytochrome. The ionic strength of the solution was controlled by using the following buffers: 1 mM E D T A plus 5 mM phosphate (pH 7.0) ( / x = 14 mM), 5 mM E D T A plus 8 mM phosphate (pH 7.0) (/x = 42 raM), 10 mM E D T A plus 20 mM phosphate (pH 7.0) (/~ = 90 mM) and x mM N a C I , 10 mM E D T A plus 20 mM phosphate (pH 7.(I) ( ~ = [90 + x ] raM). 400 and 500 M H z ~H-NMR spectra were measured at 30°C with Bruker AM400 and AM500 N M R spectrometers, respectively. Exchangeable protons were replaced with deuterium by lyophilization. The buffer was 10 or 50 mM phosphate plus NaCI (pZH 7.0). Dioxane (3.751 ppm) was used as an internal standard. The X-ray crystal structure of the Miyazaki F cytochrome used for the calculation of electrostatic maps [13] was provided by Y. Higuchi. The Hildenborough structure was generated from the Miyazaki F coordinates by amino-acid replacement and refinement using the F R O D O software package. Results

Use of EDTA vs. semicarbazide as electron donor to 5-DRF The apparent first-order rate constants for the electron transfer from 5 - D R F H " to cyt c 3 in the presence of E D T A are determined from linear plots of log delta absorbance vs time. The apparent second-order rate constant for reduction of Hildenborough cyt c 3 by 5 - D R F H " is 5.7.108 M -1 s -J (on a per heme basis) at low ionic strengths ( I = 45 to 90 mM), and the rate constant for reduction of Miyazaki F cyt c 3 is 5.3.10 ~ M -~ s -~ at • = 9 0 raM. Under these conditions (10 m M EDTA), the reduction kinetics are biphasic for both Hildenborough and Miyazaki F cyt c 3 (Figure 1A), having a slow phase which extends over 2 ms. The nature of the slow phase is uncertain. Because semicarbazide is known to give monophasic kinetics in some systems [27], it was substituted for E D T A to generate 5 - D R F H ". The efficiency of semicarbazide as an electron donor is much lower than that of E D T A , hence absorbance changes are small. Nevertheless, in the presence of 10 m M semicarbazide, the decay curve is

147 (A)

(B)

Fig. 1. Kinetic traces of the reduction 5 - D R F H at pH 7.0, 20 m M phosphate /.~M heme. Sweep time is 5 ms. (B) 10 heme. Sweep time is 2 ms. Both traces

of Hildenborough cyt c~ by buffer. (A) 10 m M E D T A , 3 m M semicarbazide, 4.5 /.tM were monitored at 569 nm.

monophasic (Fig. 1B), exhibiting only a fast phase which is complete in 0.5 ms. Plots of the observed rate constants as a function of cyt c 3 concentration both in the presence of E D T A (fast phase) and semicarbazide fall on the same line, as shown in Fig. 2. The slow phase which is observed in the presence of E D T A is presumed to be an artifact, as was reported previously [27]. E D T A rather than semicarbazide was used in all phototitration experiments because its high efficiency resulted in a good signal-to-noise ratio and the artifactual slow phase could be accurately subtracted out.

Phototitration and reduction by 5-DRFH" at low ionic strength Although the reduction of fully oxidized cyt c 3 by 5-DRFH" can be described with a single apparent second-order rate constant, the four heroes may not have identical reactivities with the reductant. Insight into the intrinsic electron transfer rate constant of the individual hemes was obtained by performing flash photolysis experiments on cyt c 3 samples which had previously been partially reduced by white light illumination. The observed pseudo first-order rate constants (kob S) obtained at I = 90 mM are plotted as a function of the percent heine reduction as shown in Fig. 3A for the Hildenborough cytochrome. The nonlinear character of the plot indicates that the second-order rate constant for reduction changes as the protein is progressively reduced; with a single second-order rate constant the plot of kobs vs. percent heine reduction should yield a straight line. There are two plausible

mechanisms which can be invoked to explain the dependence of the apparent second-order rate constant for heme reduction on the reduction state of all four hemes. In Mechanism 1, 5-DRFH" reacts with each heme group, yielding apparent second-order rate constants for reduction of the individual hemes which fall within a factor of 3 to 5 of each other and are not resolvable in the kinetic measurements (AA vs. time). In the phototitration experiment the hemes with the highest reduction potentials are reduced first, and as the titration progresses the reaction of 5-DRFH" with only the remaining oxidized (lower potential) hemes is observed. In effect, the phototitration deconvolutes the complex, overlapping kinetics of the reduction of the four heme groups. Because the four hemes are not equivalent in their reaction with 5-DRFH', the apparent second-order rate constant changes as the phototitration progresses. Alternatively, in Mechanism 2, 5-DRFH" reacts preferentially with only one or two hemes, which then can transfer electrons to the remaining hemes via rapid intramolecular electron transfer. It is thermodynamically most reasonable to expect that the intramolecular electron transfer would occur from low- to high-potential heme; thus, this mechanism would necessitate reduction of the lowest potential heme first, followed by intramolecular electron transfer to a higher potential heme. The change in the apparent second-order rate

i

i

i

i

i

O

3

0

0

,p, o X

-he

~b

2'o

3'o

4'o

10

60

Cytochrome c 3 (/~M) Fig. 2. Pseudo-first-order rate constants for the reduction o f cyt ¢3 by 5 - D R F H ' as a function of heine concentration at pH 7.0, 20 m M phosphate buffer. ©, 10 m M semicarbazide; × , from the fast phase of the reduction in the presence of 10 m M EDTA. The solid line is the least-squares fit to the observed data.

148 20,000

t

i

i

i

i

o

(A)

15,000

o

"7

Data Fit

10,000

5000

210 r

25,000

t

20,000

o

410 I

610 I

810 i

100

(a)

"7

15,000

~

o

Data - - e Fit

"~ 10,000

o

5000 I

20 [Reduced

410

I

60

8=0

100

Heine], %

Fig. 3. Pseudo-first-order rate constants for the reduction of cyt c 3 by 5-DRFH" plotted as a function of the percent heine reduced by steady-state photolysis prior to laser flash photolysis at an ionic strength of 90 mM. (A) Data for Hildenborough cyt c 3 with the theoretical curve obtained using two rate constants. (B) Data for Miyazaki F cyt c 3 fit using two rate constants. Rate constants given in Table I.

constant as the cyt c 3 becomes more reduced would then reflect a change in the reactivity of only one or two preferred hemes rather th~in all four hemes. Mechanisms 1 and 2 are kinetically indistinguishable because, in both cases, intramolecular electron transfer is rapid [8] and the intermolecular transfer of electrons from 5 - D R F H ' to the cyt c 3 heine will be rate-limiting. However, Mechanism 1 is much more appealing than Mechanism 2 based upon the following arguments. If we consider the relatively large solvent exposure of the heme groups in cyt c 3 (127 to 168 ~2, see Discussion), it is reasonable to expect that a small reductant such as 5 - D R F H , which reacts readily with the much less exposed (32 to 49 ,~2) heme groups of mitochondrial cytochrome c [31], will react with all hemes in a second-order fashion as required in Mechanism 1. Given this, it is still conceivable that reaction of 5 - D R F H " with the high-potential heme may outcompete reactions with the lower potential hemes. However, the intramolecular electron transfer from high- to lowpotential hemes would be energetically unfavorable. Thus, although Mechanism 2 cannot be discounted based upon the experiments reported here, it is viewed as much less plausible than Mechanism 1, and the

kinetic analysis presented here is based on Mechanism 1. It should be noted that this Mechanism does not exclude the possiblity of intramolecular electron transfer, indeed, intramolecular electron transfer is a welldocumented property of cyt c 3 (Ref. 9 and references included). Thus, in the initial stages of the phototitration experiment, electrons that entered the cyt c 3 molecule via the highest potential heme are transferred intramolecularly to other hemes [9]; however, the majority of the electrons remain on the highest potential heme. In order to obtain an estimate of the individual rate constants for each reactive heme, the concentration of the oxidized form of each individual heme was first determined. The amount of each heme that remains oxidized after the phototitration can be calculated using the macroscopic reduction potentials of the hemes [16]. The macroscopic reduction potentials for the Hildenborough cyt c 3 are similar to those for the Miyazaki F: - 263, - 321, - 329 and - 381 mV (Niki, K., personal communication). Having the oxidized heme concentrations, the data are then fit with the minimum number of kinetic parameters. As a single rate constant does not suffice, fits with two rate constants were made. The equation for kob.~ can be written as one of several combinations of the four oxidized heme concentrations and the two kinetic parameters: kob s = ki([Cl ] 4- [c 2 ]) 4- kii([c3] 4- [c4l ) = ki([cl]+

[ c 3 ] ) + kii([c2] + [c4])

(la) (lb)

= ki([Cl] 4- [c4] ) 4- kii([c2] 4- [c3] )

([c)

= k i([Cll 4- [c2] 4- [c3] ) 4- kii[c4]

(2a)

= k i [ C l ] + k i i ( [ c 21+[c 3]+[c4])

(2b)

= ki[£2] + kii([Cl ] 4- [c3] 4- [c4] )

(2c)

= ki[c3] + kii([c1]+[c2]+[c4])

(2d)

where ki and k~i denote the apparent second-order rate constants and c n denotes the concentration of oxidized heine n. The numbering of the heroes is based on their order of reduction potentials, with heme 1 assigned as the highest potential heine and soon [16]. For Eqn. la, for example, k i = k 1 = k 2 while kii = k 3 = k 4. The data are fit using a nonlinear least-squares approach to minimize the least-squares error with each of the equations listed above ( l a - 2 d ) . The standard deviation for the calculated rate constant is estimated by varying the rate constant in question while holding the other p a r a m e t e r constant [32]. The resulting error wells (not shown) are well-defined and symmetrical. For the reduction of Hildenborough cyt c 3, the fit having the lowest final least-squares error is obtained with eqn 2b, and corresponds to the solid line in Fig.

149 TABLE 1

8000

i

i

1

i

i

(A)

Apparent second-order rate constants for reduction of cyt c ~ by 5DRFH" 60OO

The errors reported are the range of each rate constant that results in a )0% increase in the least-squares error of the fit. Hildenborough

k~ ke k3 k4 Rate

high •(500 mM) b xl0-SM t s i

low •(90 mM) ~ ×)0 S M - I s - I

10.0±1.0 4.2±0.2 4.2±0.2 4.2+1).2

2.6+0.3 2.6+0.3 2.6±0.3 8.8±0.4

8.0±1.0 5.2±0.2 5.2±0.2 5.2±0.2

derived

o Data

4000

Miyazaki F

low •(90 mM) ~t ×10-SM t s i

constants

~sk

2000

I

I

I

I

I

0

20

40

60

80

i

i

i

i

i

100

6000

f r o m fits u s i n g E q n . 2 b w i t h k i = k I a n d

D~te

kii = k 2 = k 3 = k 4. b R a t e c o n s t a n t s d e r i v e d f r o m fits u s i n g E q n . 4 w i t h k i = k t = k e = k 3 a n d kii = k 4.

4000

3A. T h e r e s u l t i n g r a t e c o n s t a n t s a r e given in T a b l e I. A l t h o u g h t h e h e m e s do n o t r e a c t i d e n t i c a l l y with 5D R F H , the r a t e c o n s t a n t s do not differ by m o r e t h a n 3-fold. This is c o n s i s t e n t with t h e a p p a r e n t m o n o p h a s i c c h a n g e in a b s o r b a n c e o b s e r v e d with a single flash. T h e d a t a can b e fitted using t h r e e a n d f o u r r a t e c o n s t a n t s as well, b u t t h e fits a r e not significantly i m p r o v e d by using a d d i t i o n a l k i n e t i c p a r a m e t e r s , a n d so t h e least c o m p l e x m o d e l is r e p o r t e d here. F o r t h e r e d u c t i o n o f M i y a z a k i F cyt c 3 by 5 - D R F H the n o n l i n e a r i t y o f t h e kob s vs. p e r c e n t r e d u c t i o n plot (Fig. 3B) is not as g r e a t as t h a t for t h e H i l d e n b o r o u g h cyt c 3, b u t t h e fit with two r a t e c o n s t a n t s (as shown, using Eqn. 2b) still gives a significantly lower leasts q u a r e s e r r o r t h a n d o e s a fit with a single r a t e c o n s t a n t (not shown). A s a n t i c i p a t e d from t h e plot in Fig. 3B, t h e d i f f e r e n c e b e t w e e n t h e two r a t e c o n s t a n t s ( T a b l e I) is q u i t e small (less t h a n 2-fold). Typically, a d i f f e r e n c e of less t h a n 3-fold b e t w e e n any two r a t e c o n s t a n t s is difficult to d e t e c t a n d / o r m e a s u r e a n d is not conside r e d to be significant. In this instance, t h e d i f f e r e n c e s in the r a t e c o n s t a n t s r e p o r t e d in T a b l e I ( a n d T a b l e II), a l t h o u g h small, a r e c o n s i d e r e d to be significant b e c a u s e f u r t h e r s i m p l i f i c a t i o n o f the fit (i.e., r e d u c i n g t h e n u m b e r o f r a t e c o n s t a n t s from 2 to 1) results in an i n c r e a s e in fitting e r r o r o f 3 0 - 4 0 % .

2000

I

0

I

I

I

I

20 40 80 80 [Reduced Heme], %

100

Fig. 4. Pseudo-first-order rate constants for the reduction of cyt c 3 by PDQ +" as a function of percent heme reduced prior to the flash at an ionic strength of 16 mM. (A) Hildenborough cyt c 3 fit with three rate constants; (B) Miyazaki F cyt c 3 fit with three rate constants (Table I1). r a t e constant. In this case, however, the l e a s t - s q u a r e s e r r o r was significantly l o w e r e d by fitting with three, r a t h e r t h a n two, r a t e constants. A total o f seven different fits with t h r e e r a t e c o n s t a n t s a n d f o u r h e m e conc e n t r a t i o n s w e r e investigated. T h e e q u a t i o n which p r o vides the s u p e r i o r fit g r o u p s h e m e s 2 a n d 3 t o g e t h e r : kobsd = ki[cl ] + kii([c 2] + [c3]) + kiii[c4]

(3)

Fits with f o u r r a t e c o n s t a n t s d i d not give significantly lower l e a s t - s q u a r e s e r r o r s t h a n the fit using Eqn. 3. T h e resulting r a t e constants, given in T a b l e II, a r e quite similar for b o t h Miyazaki F a n d H i l d e n b o r o u g h TABLE II Apparent second-order rate constants for PDQ +" reduction of cyt c ~

Phototitration strength

and

reduction

by P D Q +" a t l o w ionic

T h e effect o f t h e e x t e n t o f r e d u c t i o n o f cyt c 3 on the a p p a r e n t s e c o n d - o r d e r r a t e c o n s t a n t for h e m e r e d u c tion was i n v e s t i g a t e d by p h o t o t i t r a t i o n e x p e r i m e n t s using a c h a r g e d r e d u c t a n t , P D Q +'. T h e o b s e r v e d p s e u d o f i r s t - o r d e r r a t e c o n s t a n t o b t a i n e d at I = 14 m M is p l o t t e d as a f u n c t i o n o f p e r c e n t h e m e r e d u c t i o n in Figs. 4 A a n d 4B for H i l d e n b o r o u g h a n d M i y a z a k i F cyt c 3, respectively. A s was t h e case for t h e 5 - D R F H " e x p e r i m e n t s , the d a t a c a n n o t b e d e s c r i b e d with a single

Hildenborough

kI k2 k3 k4

Miyazaki F

low I(16 mM) ~ ×10 S M - I s a

high •(500 mM) b ×10-8M Is-1

low 1(16 mM) a ×10 SM Is - j

1.6 ±0.2 0.60±0.06 0.60±0.06 1.6 ±0.1

1.4±0.1 1.4±0.1 1.4±0.1 3.3±0.2

1.1 ±0.1 0.60±0.04 0.60±0.04 1.9 ±0.1

R a t e c o n s t a n t s d e r i v e d f r o m u s i n g E q n . 3 w i t h k i = k l , kii = k 2 = k 3 a n d kii i = k 4. b R a t e c o n s t a n t s d e r i v e d f r o m fits u s i n g E q n . 4 w i t h k i = k I = k 2 = k 3 a n d kii = k 4.

150 cyt c3, with values for k 2 and k 3 (both = kii in Eqn. 3) lower than those for k I ( = k i) and k 4 ( = kii i) by 2- to 3-fold. In comparing Tables I and II it is noted that, in general, the rate constants obtained with P D Q + relative to those for 5 - D R F H " alone are significantly lower. This is not unexpected, since the driving force with P D Q +" is app. 100 mV less than with the 5 - D R F H " species.

Ionic Strength Dependence 0

O

Effect o f ionic strength on cyt c 3 heme reduction In order to determine the electrostatic environment surrounding the cyt c 3 heine groups, the ionic strength dependence of the second-order rate constant for reduction of cyt c 3 was examined. Previous measurements for the reduction of Hildenborough cyt c 3 by MV +' generated by pulse radiolysis [33] have indicated the presence of a strong ionic strength dependence. In the flash photolysis experiment reported here, MV + reduction of Hildenborough cyt c 3 is monophasic in all cases, and the apparent second-order rate constants (obtained from the concentration dependence of the observed pseudo-first-order rate constant, similar to the data shown in Fig. 2) are listed in Table III and plotted in Fig. 5. The apparent second-order rate constant increases with increasing ionic strength, consistent with a plus-plus electrostatic interaction between MV + and cyt c a. At the lower ionic strength, a smooth curve is observed. Using the parallel-plate electrostatic model [5], which has been successfully applied to a large number of cytochromes, the five data points at lower ionic strengths are fit as described previously [34]. The calculated curve for this model is shown as a solid line in Fig. 5. The effective charge on the interaction domain of Hildenborough cyt c3 is estimated from this as + 2.5 by analogy to mono-heme cytochromes c [5], using an effective dielectric of 50 and a radius of 4.5 A for the interaction domain. The electrostatically corrected rate constant (k=) and electrostatic interaction energy (Vii) are 1.9-10 s M -1 s -1 and 1.1 k c a l / m o l , respectively. Between I = 250 and I = 500 m M a transition occurs, and the apparent second-order

TABLE III Apparent second-order rate constants for reduction of fully oxidized Hildenborough cyt c 3 by M V +^ in the presence of EDTA Ionic strength

k × 10 - s M - 1 s - 1 (mM)

16

0.86

43 92 163 254 500 723 1000

1.10 1.25 1.33 1.41 1.71 2.09 2.43

E

0

I

0.5

I

1.0

I 1/2

Fig. 5. Ionic strength dependence of apparent second-order rate constant for reduction of Hildenborough cytochrome c 3 by MV ÷'. The solid line represents the best fit curves obtained by non-linear least-square fitting to the five observed points at lower ionic strength. Rate constants given in Table III.

rate constant continues to increase with increasing ionic strength. At the higher ionic strengths (ie., I --- 500 raM) the electrostatic contribution to the rate constant for reduction by P D Q + following phototitration is anticipated to be minimized. If so, the reduction of cyt c 3 by P D Q +" should become similar to the reduction of cyt c 3 by the electrostatically neutral 5 - D R F H ', except for the difference in driving force. The data for reduction by both species at I = 500 mM (Fig. 6) are best fit using two rate constants: kob s = k i ( [ C l l + [ c 2]+ [C3])+ kii[c 4]

(4)

The P D Q +" reduction at high ionic strength is, as anticipated, similar to the 5 - D R F H " reduction, with k a > k~ = k : = k 3 where k 4 is 2- to 3-fold greater than the other rate constants (Tables I and II, high I). The dependence of the rate constant for cyt c 3 reduction on the percent heme reduction (for Hildenborough cyt c 3) at I = 500 mM (Fig. 6) is distinctly altered from the data at I = 90 to 16 m M (Figs. 3A and 4A, respectively) for both 5 - D R F H " and P D Q +'. Although the change in rate constant for any given heme in going from low to high ionic strength is only 2- to 3-fold, the overall relationship of all four rate constants relative to one another has been significantly altered. Thus, for 5 - D R F H reduction of Hildenborough cyt c 3

151 16,000

I

I

I

I

o

(A)

"~,~_

12,000

the pattern of a high k~ with k 2 = k 3 = k 4 at low ionic strength shifts to a pattern of a high k 4 with k 1 = k 2 = k 3 at high ionic strength. The pattern for the P D Q ÷ reduction of Hildenborough cyt c 3 likewise changes with increasing ionic strength, from k~ = k 4 > k 2 = k 3 to k 4 > k 7 = k 2 = k 3.

I

o Data F it

'OR

8000

°\o

4000

I

I

I

I

I

0

20

40

60

80

I

I

I

I

I

(8)

o

6000

100

o~o °

o

~

o

--

Data Fit

4000

g 2000

I

I

I

I

I

0

20

40

60

80

100

[Reduced Heine], %

Fig. 6. Pseudo-first-order rate constants for the reduction of Hildenborough cyt c 3 by (A) 5-DRFH" and (B) PDQ ÷ at an ionic strength of 500 mM. Solid lines are the best fits obtained by non-linear least squares fitting using two rate constants (Tables I and II).

°.2 t H

0.4

e

/HemeI

0

~

~

fl

,

, F

-

~

:

--0.2

G B

0.2 I Heme III C 0l

--0.2

*-

- -

-

;

I

0.2] /

--0.21

0

"

:.1

D

' Heme II

J

'

0.5 [NaCI], M

1.0

Fig. 7. The change in chemical shift of the heme methyl resonances from a series of 1H-NMR spectra of Miyazaki cyt c 3 at 30°C with increasing [NaCI]. Heme methyl resonances were identified according to Fan et al. [9].

Ionic strength-dependent 1H-NMR The ionic strength dependence for the reduction of cyt c3 by the electrostatically neutral 5 - D R F H suggests that a change in the structural properties or reduction potential of one or more hemes of cyt c 3 occurs at 90 mM < I < 500 mM. Hence, the molecular species of cyt c 3 present at low ionic strengths ( < 90 mM) may be different from those at higher ionic strengths ( > 500 mM). Therefore, the effect of ionic strength on the =H-NMR spectra of Hildenborough and Miyazaki F cytochromes were examined to determine whether changes in the redox-sensitive heme methyl resonances could be detected. The chemical shift changes of the heme methyl groups of Miyazaki F cyt c 3 were plotted as a function of NaC1 concentration in Fig. 7. Similar changes were observed for Hildenborough cyt c 3. The chemical shift of some of the heme methyl signals is, in fact, ionic strength dependent, indicating an alteration in heme environment. The most pronounced changes in the N M R resonances are observed for the two highest-potential hemes (hemes 1 and 4), with the two lower potential hemes (hemes 2 and 3) relatively unaffected. Finally, it should be noted that no change in the visible absorption spectrum of cyt c 3 was observed over the range of ionic strengths studied. Discussion Previous studies of electron transfer to c 3 cytochromes from small molecules with very low reduction potentials, such as dithionite and MV +, have shown that heme reduction occurs readily in the absence of oxygen, with rate constants in the range of 108 M -1 s -1 [35-37]. As reported here, reduction of Miyazaki F and Hildenborough cyt c 3 by 5 - D R F H , P D Q +" and MV ÷" is observed with rate constants in the 107-108 M -~ s = range as well. The kinetic profiles for reduction of the fully oxidized cyt c 3 do not indicate the complexity of 4 macroscopic and 32 microscopic reduction potentials: a single kinetic process is observed. It is only when the partially reduced cyt c 3 species is generated, as done in this study, that differences in heme reactivity can be observed.

Steric contributions to reduction of cytochrome c 3 Many factors control h e m e reduction potential, and it is generally not possible to draw a correlation between a single factor and reduction potential that

152 carries general significance for a large number of heme proteins [38-40]. However, it is well established that biological electron transfer is an outer sphere process which can be described by Marcus theory [5,22,41,42]. For cytochromes, the semiempirical Marcus equation applies: AG ~ = zaG°+ (zlG*(0)/In 2) ln{l + exp(zlG ° In 2/AG~(0))}

zlG*'/RT)

k,: t = ~,~, exp( -

(5) (6)

Here zIG* is the activation free energy, zIG ° is the driving force of the reaction resulting from the difference in oxidation-reduction potential between cyt c 3 and the reductant used ( = -nFzIEm), AG*(O) is the intrinsic barrier, and vet is a frequency factor reflecting the limiting electron transfer rate constant. The intrinsic barrier AG*(0) is equal to A/4, where A is the reorganizationai energy. The rate constant for the rate-limiting electron transfer step, ket , is not directly measured in the experiments reported here. Rather, the apparent second-order rate constant is determined which is the product of ket and the equilibrium constant for the formation of a transient reductant-cytochrome complex prior to electron transfer [22]. Thus, the apparent second-order rate constants measured

1010

"]

O3

i

I0~

//

/

t

t

D/ /

/

~

// ,.o

/

I/i II

10 e

/

/ // , /// / I/ //

/

zx H!gh Potential c y t c n M!yazaki cyt c 3 0 Hild cyt c 3

/// /

10 7

0.0

t

i

I

i

0.2

0.4

0.6

0 8

1.0

delta E m, V Fig. 8. The second-order rate constants for the reduction of cyt c 3 and high-potential cytochromes c by 5-DRFH" as a function of the difference between the macroscopic reduction potentials of the cytochromes and 5-DRFH . The solid line is the best fit obtained by non-linear least square fitting to the data using Eqns. 5 and 6. Data for the high potential heroes are reploned from Ref. 31, Fig. 1, cytochromes D, E, F, H, J, L, which correspond generally to cytochromes c2, c-553, c-551, c-555 and c-550. The dashed lines yield + 10% the values of v~t and A that give the best fit.

here (ie., k 1, k 2, k 3 , and k 4 ) a r e simply referred to as as kobs. The value for kob s, based upon Eqns. 5 and 6, therefore depends upon a number of factors including the difference in oxidation-reduction potential (AE m) distance and intervening media, as well as orientation and conformational energy which are reflected in vet and A. Thus, within a family of structurally homologous redox proteins, Eqns. 5 and 6 should give a plot of In k,,b~ vs. AE m which yields a smooth curve described by A and Vet. In Fig. 8 the observed rate constants for the reduction by 5-DRFH" are plotted against ZIEm along with previously reported data for high-potential cytochromes c [27]. The least squares best fit of Eqns. 5 and 6 to these data yields a value of 14.4 kcal/mol (0.62 eV) for A and 3.5.109 M - l s-1 for uet. Values for kobS for both Class 1 and Class 2 cytochromes fall within + 10% of the fit to the Marcus semiempirical equation, demonstrating a correlation between kob., and A E m over a wide range (700 mV) of reduction potentials. This fact indicates that the essential mechanism of the electron transfer from 5-DRFH" is the same for these c-type cytochromes, in spite of their wide variety of structures and properties. This is an unexpected result, given the fundamental differences in structure and properties between the Class I and Class III cytochromes [5]. Steric and electrostatic effects which are not accounted for by Eqns. 5 and 6 have been shown to cause substantial deviations from the expected curve in other redox systems [5,22], and probably contribute to the scatter seen in Fig. 8 as well. As an example of this, note that the highest rate constant for the 5 - D R F H reduction of Hildenborough and Miyazaki F cyt c 3 at low ionic strength is that for the heine with the highest reduction potential. The rate constants for the remaining hemes are essentially equal to one another, in spite of the significantly lower redox potential of one of the hemes. The observed k ob~, value for the lowest reduction potential heme, Heme II, is therefore higher than expected based upon driving force. It is known that the cyt c 3 hemes exhibit much more exposure to solvent (127 to 168 ,~2) than do the single hemes of the mitochondrial cytochromes c (32 to 49 ,~2) [13]. This implies that the large solvent accessibility of Heme II relative to the other hemes results in an increased reactivity, and that a limit has been reached in terms of the ability of a lowered reduction potential to compensate for an increased solvent exposure.

Electrostatic contributions to reduction of cytochrome c s In addition to the apparent influence of solvent accessibility, electrostatic effects may contribute to the electron transfer properties of cyt c 3, considering that the Miyazaki F and the Hildenborough cytochromes c 3 are positively charged at pH 7, having p l values of 10.5

153 [43]. The rate constants for reduction of cyt C 3 by 5-DRFH, an electrically neutral species, should be independent of ionic strength and thus useful as a control. However, these rate constants were found to be sensitive to ionic strength: at high ionic strength the lowest reduction potential heme, Heme II, has the largest rate constant for reduction, and the kobs for the highest reduction potential heme, Heine IV, has been decreased by 3-4-fold. It is known from the X-ray structure that Miyazaki F cyt c 3 has an asymmetric charge distribution, with much of the positive charge localized close to the highest potential Heme IV [13]. To obtain a more quantitative view of the surface charge distribution, electrostatic fields were calculated using the modified

Tanford-Kirkwood method of Matthew [44,45]. These fields are displayed as stereo views in Fig. 9. Calculation of the electrostatic fields was also carried out using a finite difference algorithm to solve a linearized Poisson-Boltzmann equation (DelPhi package, Biosym Technologies [46]), with results not significantly different from the maps shown in Fig. 9. The fields from the positively charged residues are more extensive than those from the negatively charged residues for both proteins, as expected. The positive charges originate solely from lysine residues, with the exception of the amino terminal nitrogen and, in the Hildenborough protein, a single arginine residue. In the Miyazaki F cyt c 3 (Fig. 9A), some positive charges in the region of the amino terminus are seen to the upper left of Heme I.

(A)

(B)

Fig. 9. Stereo views of the calculated electrostatic fields for Miyazaki F (A) and Hildenborough (B) cytochromes c 3. The four h e m e groups and the carbon alpha backbone are displayed in addition to the negative fields (dashed lines) and positive fields (solid lines). The heroes are, clockwise from the upper left, I, II, IV and III. The electrostatic maps were calculated at an ionic strength of 0.1 M and an energy level of 2 kT. The graphics are generated by F R O D O software and are viewed using an Evans and Sutherland Model PS390 graphics display.

154 These charges do not appear to be close to any of the four hemes. The bulk of the positive charge is located in the vicinity of Heme IV, and is due to lysine residues located at positions 15, 57 and 58 (pictured here to the right of Heme IV) as well as in the region of the C-terminus at positions 72,94,95 and 101. The pattern of charge is very similar for the Hildenborough cyt c 3 (Fig. 9B) with the exception that the positive fields overlay one another to a greater degree than in the Miyazaki F. Of particular interest are the fields close to Heme IV, where an additional lysine at position 102 in the Hildenborough cyt c 3 increases the electrostatic potential noticeably. The ionic strength sensitivity of the rate constants to reduction by a neutral species such as 5-DRFH" may be attributed to a change in reduction potential with change in ionic strength. A change in reduction potential of the hemes of cyt c 3 could be the result of a conformational change in the protein or, may result from a decrease in the magnitude of the electrostatic fields as ionic strength is increased. These potential fields either partially or wholly envelope the hemes, and altering the size of the field would change the local electrostatic potential of the heme Fe. Although the magnitude of electrostatic interactions between buried heme groups is, in general, not well understood, there is evidence that ionizable groups such as heme propionates can influence heme reduction potentials [36,47,48]. The relatively close positioning of the four heine groups in cytochrome c3,with iron-iron distances ranging from 11.0 ,~ to 17.8 A [13], strongly suggests that the electrostatic field due to the positive charge on the heme iron of one heme may influence the electrostatic potentials of the other heine groups. In addition to the unexpected effects of ionic strength on the kinetics of cyt c 3 reduction by a neutral species, an unusal ionic strength dependency is also observed for cyt c 3 reduction by the postively charged MV ÷. In this case, the increase in the apparent second-order rate constant for reduction with increasing ionic strength does not saturate, but is discontinuous, continuing to increase even at an ionic strength of 1 M. The lack of an apparent saturation of the second-order rate constant at high ionic strength has not been previously observed in studies of reduction kinetics of a wide variety of c-type cytochromes, high potential iron-sulfur proteins and copper proteins (reviewed in Ref. 5). It is important to note that the reduction potentials of the cyt c 3 hemes at high ionic strength have not been reported, and, in addition to possible changes in reduction potential, the assignments of redox potential to heine may be altered as well. The rate constants calculated at high ionic strength given in this report (Tables I and II) could only be determined with available reduction potential data, and are therefore valid

only if the reduction potentials have not changed significantly at high ionic strength. Thus, the dependence upon ionic strength of the reduction potentials must be determined before the kinetic data at high ionic strength can be definitively characterized. In the case of reduction of cyt c 3 by a charged species, PDQ ÷', at low ionic strength, the kob s for the lowest potential heme, Heme II, is higher than anticipated. In fact, for both the Miyazaki F and Hildenborough cytochromes c3, the Heme II kobs is not only higher than that for the intermediate reduction potential hemes, it is as great as or greater than the kob~ for the highest potential heme. This becomes understandable when the positively charged environment of the highest-potential heme is considered. This should result in a lower kob~ for reduction by a positively charged species such as PDQ ÷. The large k obs for the lowest potential heme is consistent with its large solvent accessibility and its largely neutral electrostatic environment which, unlike the protein surface at the highestpotential heme, will be non-repulsive to PDQ +'. At high ionic strength, the relative rate constants for PDQ ÷" reduction of the Hildenborough cyt c 3 are substantially altered from the pattern observed at low ionic strength. Rate constants for all heroes are essentially equal, with the exception of the lowest potential heme, which is larger by more than 2-fold (assuming that the lowest potential heine is still Heme I1 at high ionic strength). At this high ionic strength the relative rate constants for 5 - D R F H reduction of cyt c 3 are very similar to those for the reduction by PDQ ÷'. Therefore, the contribution of electrostatic interactions between the reductant and cyt c 3 surface charges must be suppressed to a great extent, with the values of kob s primarily reflecting the accessibility of the reductant to the heme. Given the discussion presented to this point, the high-ionic-strength data for both 5-DRFH" and PDQ +" can now be used to illustrate the effects of sterics and electrostatics, when viewed in the following manner. At high ionic strengths steric factors predominate over electrostatic factors in their influence o n kob s. As ionic strength is decreased, electrostatic effects are added to the still present steric factors. For the neutral reducrant, the kob s for Heine IV (highest potential, positive electrostatic field) increases 4-fold. N M R data indicate that the heine environment has been altered. For the charged reductant, the expected decrease in kob s does not occur. For this heine, the plus/plus electrostatic repulsion of the reductant and the protein is overshadowed by another, perhaps steric, effect. For Heme II (lowest potential, neutral electrostatic field), the kobs is reduced 2-fold by the addition of electrostatic effects, whether the reductant is charged or not. Again, the reductant charge does not appear to be an important factor. The effect of adding charge to

155 the reductant does, in fact, exist, and is clearly demonstrated at low ionic strength. Therefore, as a prerequisite for distinguishing electrostatic from steric effects in these cytochromes, the properties of the species present at high ionic strength, such as reduction potential and structure, must be evaluated.

Summary The studies reported here result from the application of phototitration techniques to discriminate among the spectrally equivalent heroes in the multiheme cytochrome c 3. This approach has permitted the measurement of individual rate constants for this system, establishing that the multiple hemes of the Desulfovibrio vulgaris Miyazaki F and Hildenborough cytochromes c 3 vary in their reactivity with small, low potential reducing agents, with both steric and electrostatic factors contributing to the process of electron transfer. Analysis of the resulting rate constants has provided the first evidence that the Marcus theory of outer-sphere electron transfer applies to the Class 3 cytochromes. Cytochromes from both the Miyazaki F and Hildenborough strains are very similar in terms of their reactions with exogenous reductants, as expected from their amino acid sequence homology. The apparent second-order rate constants for reduction are consistent with the previously reported assignments of reduction potential to the individual heroes made by NMR. The unusual ionic strength sensitivity of the rate constants indicates that electrostatic interactions are important to the structure a n d / o r to the heine reduction potentials of cyt c 3.

Acknowledgements This work was supported in part by the Office of Naval Research (M.A.C.) and by a fellowship from the Ministry of Education and Culture of Japan (H.A.). We are grateful to James T. Hazzard, Gordon Tollin and Terry Meyer at the University of Arizona for their valuable discussions, to Gordon Tollin and Mark Walker for generously supplying 5-deazariboflavin and PDQ 2÷, and to James B. Matthew for providing Fortran code for the electrostatics calculations.

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