Properties and Recent Insights - American Chemical Society

22 Cytochrome c : Properties and Recent Insights

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3

M . A. Cusanovich , J. H . Hazzard , and G . S. Wilson 1

1 2

1

2

Department of Biochemistry, University of Arizona, Tucson, ΑΖ 85721 Department of Chemistry, University of Kansas, Lawrence, KS 66045

The cytochromes c from the sulfate-reducing bacteria of the genus Desulfovibrio comprise a class of c-type cytochromes that are distin guished from the more familiar mitochondrial cytochromes c in two important respects. First, cytochromes c contain four heme groups, rather than one, bound to a single polypeptide of less than 120 amino acid residues. In addition, the redox potentials of the multiple hemes are very low ( — 165 to —400 mV) in contrast to +260 mV for mitochondrial cytochrome c. Importantly, the electrochemical and electrical properties of the cytochromes c are unique among biologi cal redox proteins. The cytochromes c modify electrodes in a way that facilitates heterogeneous electron transfer and in dry films have the ability to change resistivity by up to 10 orders of magnitude in going from the fully oxidized to fully reduced states. The structural properties, oxidation-reduction potentials, heterogeneous and homo geneous electron-transfer kinetics, electrical properties of protein films, and the utilization of site-directed mutagenesis in understanding the structure and function of the cytochromes c are discussed. The focus is on new information and insights that further our understanding of the cytochromes c as well as define the issues that must be addressed to exploit fully this important and interesting protein. 3

3

3

3

3

3

THE

CYTOCHROMES

C

3

A R E C L A S S III

C - T Y P E C Y T O C H R O M E S (1)

that w e r e

originally isolated i n 1954 f r o m Desulfovibrio (2, 3 ) . A l t h o u g h widely dis tributed amongst the Desulfovibno, cytochromes c have only recently b e e n f o u n d i n other species; for example, i n the p u r p l e phototrophic bacterium H 1 R ( T . E . M e y e r , personal communication) a n d i n Desulfuromonas acetoxi3

0065-2393/94/0235-0471 $08.00/0 © 1994 American Chemical Society

In Biomembrane Electrochemistry; Blank, M., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

472

BIOMEMBRANE ELECTROCHEMISTRY

dans ( 4 ) . F u n c t i o n a l l y , i n the Desulfovibno,

the cytochromes c

3

mediate

electron transfer between hydrogenase a n d the i r o n - s u l f u r p r o t e i n ferredoxin, as shown schematically ( 5 )

hydrogenase ^ cytochrome c Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 10, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch022

3

^ ferredoxin

(1)

R e d u c e d ferredoxin reacts w i t h proteins that participate i n the dissimilatory reduction o f sulfate to sulfide; oxidized ferredoxin reacts w i t h pyruvate dehydrogenase that catalyzes the conversion o f pyruvate to acetyl C o A (phosphoroclastic reaction). I n sulfate reduction, molecular hydrogen is the electron source, a n d i n the phosphoroclastic reaction, protons are the t e r m i nal electron acceptor a n d hydrogenase mediates electron transfer between cytochrome c a n d protons o r molecular hydrogen. 3

C y t o c h r o m e c is distinct f r o m the class I c-type cytochromes (for example, m i t o c h o n d r i a l cytochrome c) a n d class II ο type cytochromes (for example, cytochrome c') i n terms o f out-of-plane ligation. T h e class I I I cytochromes have histidine i n b o t h the fifth a n d sixth h e m e coordination positions versus his-met (class I) o r his-vacant (class II) a n d also differ i n the n u m b e r o f covalently b o u n d h e m e groups p e r peptide chain: four h e m e groups for class I I I versus one h e m e group for classes I a n d I I . M o r e o v e r , the cytochromes c have a n u m b e r o f u n u s u a l properties that make t h e m valuable experimental materials f o r understanding biological electron trans fer. 3

3

T o date, the cytochromes c f r o m four species have b e e n extensively studied i n terms o f b o t h structural a n d physicochemical properties. These species are cytochrome c f r o m Desulfovibno vulgaris M i y a z a k i (Miyazaki), Desulfovibno vulgaris H i l d e n b o r o u g h ( H i l d e n b o r o u g h ) , Desulfovibno desulfuricans N o r w a y (Norway), a n d Desulfovibno gigas (Gigas). T h e structural properties, o x i d a t i o n - r e d u c t i o n potentials, site-directed mutagenesis, electri cal properties, a n d homogeneous a n d heterogeneous redox kinetics o f cy tochrome c w i l l b e discussed, a n d the prospects f o r future insights w i l l b e described. 3

3

3

Structure T h e amino acid sequences o f four examples o f cytochromes c ( M i y a z a k i , H i l d e n b o r o u g h , Gigas, a n d N o r w a y ) are presented i n F i g u r e 1. A n u m b e r o f features are notable. A l t h o u g h there is substantial a m i n o a c i d sequence homology between H i l d e n b o r o u g h a n d M i y a z a k i cytochrome c (85%), there is m u c h less h o m o l o g y a m o n g the other cytochromes c . F o r example, N o r w a y a n d M i y a z a k i cytochrome c have only 3 5 % sequence homology. 3

3

3

3


22.

CUSANOVICH E T AL.

Cytochrome c : Properties and Recent Insights 3

T h e amino acid sequence identity matrix f o r cytochromes sulfovibno

c

3

473

f r o m six De-

species are as follows:

• Gigas • 53 Miyazaki Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on October 10, 2015 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch022

• 5 3 85 H i l d e n b o r o u g h • 31 39 37 D. desulfuncans

E l Agheila

• 32 39 37 67 D. saleigens B r i t i s h G u i a n a • 30 35 34 40 44 N o r w a y

T h e percent identity i n amino a c i d sequence is given using the insertions a n d deletions shown i n F i g u r e 1. O n l y a small n u m b e r (25) o f sequence positions are conserved a m o n g the four sequences shown. H o w e v e r , 16 o f these 25 are associated w i t h h e m e b i n d i n g (cys a n d his); hence only nine side chains are conserved. T h i s lack o f amino acid sequence homology among the cytochromes c as a family allows f o r substantial differences i n physicoehemical properties w h i l e retaining structural homology. 3

F i g u r e 1 also provides i n f o r m a t i o n o n h e m e attachment. T h e h e m e n u m b e r i n g corresponds to h e m e designation i n the Miyazald cytochrome c crystal structure. F o r t w o o f the four hemes (heme I I I a n d h e m e II), the heme b i n d i n g sequence is cys f o l l o w e d b y two amino acids a n d then c y s - h i s . T h i s b i n d i n g sequence is typical o f e-type cytochromes: T h e histidine serves as one o f the axial ligands. H o w e v e r , the other t w o hemes have the unusual sequence cys f o l l o w e d b y four, rather than two, amino acids and then c y s - h i s . M o r e o v e r , it is quite apparent that the distal histidine, because o f its distance f r o m the h e m e b i n d i n g site i n the amino a c i d sequence ( F i g u r e 1), plays a key role i n d e t e r m i n i n g the three-dimensional structure o f the class I I I cytochromes. 3

A s far as electrostatics are concerned, Gigas a n d N o r w a y cytochromes c are acidic ( p i = 5.2) ( 6 ) a n d neutral ( p i = 6.9) ( 7 ) , respectively, whereas M i y a z a k i a n d H i l d e n b o r o u g h cytochrome c are basic ( p i = 10.5) ( 6 ) . These electrostatic properties result f r o m a net apoprotein charge o f + 8 , + 8 , + 4, a n d + 4 f o r M i y a z a k i , H i l d e n b o r o u g h , N o r w a y , a n d Gigas cytochrome c , respectively. B a s e d o n amino a c i d composition sequence a n d the fact that the four hemes provide eight propionates, w e c a n infer that at least four propionates should b e i o n i z e d at a p H 7.0 to y i e l d the observed isoelectric points, assuming that some o f the uncertainties i n the Gigas cytochrome c sequence are acidic. It is also notable that r e d u c t i o n o f all four hemes o f the cytochrome c is accompanied b y a p r o t e i n net charge decrease o f 4 because the f o r m a l heme i r o n charge w i l l change f r o m + 1 to 0. T h i s last observation suggests that ionic strength c o u l d have a very large effect o n the redox 3

3

3

3

3


474


M H G


N

A

M H G

Ρ Ρ L Q

N

A Ρ G

D

1 10 Α Ρ Κ A Ρ A D G L Κ Α Ρ Κ A Ρ A D G L Κ V D V Ρ A D G A Κ G M Κ D Y V I S A Ρ E

D

20 V V F N V V F N V V F Ν Τ V Ρ F Ρ

- - K

Η Η Η Η

s s s τ

Τ Τ Τ Κ

H H H H

K K K A

A S D Τ

-

V V V V

K K K E

30 C C C c

Τ

M H G N

Y Y Y V

Q R A K

K K G K

c c c c

- - - - - - - -

Y Y w L

-

Y Y Y V E

-

H H K S

A V V A

70 M Η M Η V Η F Η

D K E A D F I A G G K Ρ K G D K Ρ

D D B Q

C C C C

-

A G Τ Τ

Τ Τ Τ Τ

50 A G A G D G S G

C C C C

H H H H

D D Ν D

Ν S I S

M M L L

- -- -

D D D Ε F R

A A K D

A A E K

K K L K

80 D κ G Τ K F K s C V G C D κ Ν Τ K F K _ s C V G C D Α Κ G G A K Ρ τ C I S C Τ Q C I D C

-

1.00 K E L Τ G C K G S K C K D L Τ G C K K S K c K K L Τ G C K G S A c G K c Τ G Ρ Τ A C

K K K Ρ

Κ Q Κ E Κ Ν Α L

H H H H

Ρ Ρ Ζ Χ

V V Ρ X

N N G A

40 G Κ G Κ B Κ D G

Ε D Ε D Q G A

K K A K

60 D K D K D K A Ν

S S S A

A A V K

Κ Κ Ν D

G G S I K

H L E Τ A H V E V A H K D Κ A H A L

G G G K

90 Α D Α D D D Κ K

H H H H

-

K K K D

-

HEME I I

—HEME IV M H G H

Τ Τ Ε G

• HFMF T T T

Γ

M H G N

G G Β V

M M I A

-

H H H H

-

S E Ρ S Τ Τ N

HEME I

1

Figure 1. Cytochrome c amino acid sequences. Amino acid sequences are given for Miyazaki (M), Hildenborough (H), Gigas (G), and Norway (N). Numbering is for Miyazaki cytochrome c . Heme labeling is consistent with assignments from the Miyazaki cytochrome c three-dimensional structure (Figure 2). 3

3

3

potential o f the cytochrome

c

3

hemes a n d that partial reduction s h o u l d

greatly influence subsequent reduction steps. T h e three-dimensional structures o f the four cytochromes described here are k n o w n (8-10; cytochrome c

3

N . Yasuoka, personal communication). T h e M i y a z a k i

X - r a y structure is solved to the highest resolution (1.8 A ) ( 8 ) .

Because the cytochromes c

3

are structurally homologous, only the M i y a z a k i


22.

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Cytochrome c : Properties and Recent Insights

475

3

cytochrome c structure w i l l b e discussed i n detail. F i g u r e 2 presents three different stereo representations o f the M i y a z a k i structure. I n the top panel, a r i b b o n diagram shows the backbone folding a n d h e m e positioning, w h i c h establishes the relative orientation o f the four hemes a n d their relation to the molecular surface. T h e h e m e n u m b e r i n g is consistent w i t h that o f F i g u r e 1 and w i l l b e used throughout this discussion. A space-filling representation i n the m i d d l e panel o f F i g u r e 2 demonstrates the exposure o f h e m e I I ( i n green) to solvent. T h e axial histidine ligands (magenta) are clearly visible. H e m e s I a n d I V are partially visible o n the left a n d right, respectively. Oxygen atoms o f glutamic acid a n d aspartic acid residues are identified b y r e d (heme propionate oxygens are p i n k ) a n d lysine ( N Z ) b y b l u e . Some o f these residues participate i n salt bridges or i n hydrogen b o n d i n g and, thus, do not contribute to the surface electrostatic field, w h i c h is shown i n the b o t t o m panel. T h i s representation illustrates the electrostatic fields that w o u l d b e experienced b y an approaching reactant at an i o n i c strength o f 0 . 0 1 M a n d an energy o f 2 k T , a n d is calculated using the m o d i f i e d T a n f o r d - K i r k w o o d m e t h o d o f M a t t h e w ( I I , 12). T h e positive fields are blue, a n d the negative fields are r e d . A n u m b e r o f structural features are notable. T h e i r o n - t o - i r o n distances vary f r o m 1 7 . 8 to 1 1 . 0 A , a n d the angles between the h e m e planes vary f r o m 2 2 to 8 9 ° (Table I) ( 8 ) .


3

As can be seen i n the top p a n e l o f F i g u r e 2 , there is n o obvious regular relationship a m o n g all four hemes, although interheme planar angles o f near 9 0 ° ( 8 0 - 8 9 ° ) are most c o m m o n (Table I). T h e exposure o f each o f the hemes to solvent i n terms o f accessible surface area is 1 2 7 , 1 6 8 , 1 3 6 , a n d 1 3 6 A for hemes I - I V , respectively ( 8 ) . T w o points are apparent. F i r s t , relative to the class I c-type cytochromes w h e r e the accessible surface area is typically 3 2 - 4 9 A , the cytochrome c h e m e exposure is quite large. Second, h e m e I I is substantially m o r e exposed than the other three hemes, w h i c h suggests that it m a y have some u n i q u e properties. I n terms o f electrostatics, it is quite apparent that the M i y a z a k i cytochrome c has an asymmetric charge distribu tion a n d the region i n the vicinity o f heme I a n d the C - t e r m i n u s has a large positive field. T h i s asymmetric electrostatic field m a y play an important role i n the functional a n d electrical properties o f the cytochrome c as w e shall discuss later. L y s i n e residues at positions 1 5 , 5 7 , 5 8 , 7 2 , 9 4 , 9 5 , a n d 1 0 1 make up m u c h o f the electrostatic field i n the h e m e I a n d C terminus region. Reference to the amino acid sequences given i n F i g u r e 1 shows that positive charges at positions 1 5 , 5 7 , 9 5 , a n d 1 0 1 are conserved a m o n g M i y a z a k i , H i l d e n b o r o u g h , a n d Gigas cytochrome c , w h i c h suggests that the asymmet rical charge distribution is also conserved. T h i s feature persists despite the fact that M i y a z a k i a n d Gigas cytochrome c have net p r o t e i n charges o f opposite sign. I n contrast, a comparison o f M i y a z a k i a n d N o r w a y cytochrome c sequences yields conserved lysines only at positions 1 5 a n d 5 8 . T h u s , the positive field observed for the M i y a z a k i cytochrome is absent i n the N o r w a y cytochrome. Indeed, the calculated electrostatic m a p f o r the N o r w a y cy2

2

3

3

3

3

3

3


BIOMEMBRANE

ELECTROCHEMISTRY


476

Figure 2. Stereo views of Miyazaki cytochrome c showing the C backbone folding pattern (top panel), the space-filling view of the relative solvent exposure of heme II (middle panel), and the distribution of the electrostatic fields on the protein surface (lower panel). The views in all three panels are of the same x, y, ζ orientation as displayed on an Evans and Sutherland model PS390 graphics system using software packages Insight (top and middle) and Frodo (lower). 3


22.

CUSANOVICH E T AL.


477

Table I. Iron-Iron Distances and Heme-Heme Angles for Miyazaki Cytochrome c 3

Heme

I


I II

73°

III IV

22° 80°

II

III

TV

16.4 A

17.8 A

12.0 Â

12.2 A

15.8 Â 11.0 A

88.7° 59.1°

80.3°

NOTE: The upper right quadrant gives F e - F e distances. The lower left quadrant gives angles between heme planes. The heme nomenclature is that used in Figures 1 and 2 . SOURCE: Data are taken from reference 8.

tochrome is also asymmetric; all four hemes are i n a negative field, a n d only a small positive field remains at the C terminus. In summary, the cytochromes c are structurally homologous a n d have a compact structure w i t h little secondary structure. Clearly, the folding is dictated b y the presence o f the four six-coordinate hemes. I n general, the cytochromes c have an asymmetric charge distribution, a n d a l l four hemes are substantially m o r e exposed to solvent than i n the m o r e typical class I h i g h potential c-type cytochromes. 3

3

Oxidation-Reduction Potentials A characteristic o f the cytochromes c is a very l o w o x i d a t i o n - r e d u c t i o n potential. M o r e o v e r , it is obvious f r o m the m u l t i h e m e nature o f these cytochromes that the redox properties s h o u l d b e complex. I n the simplest situation, four individual redox potentials c o u l d b e expected, one f o r each h e m e . I n addition to the axial ligands o n the hemes, a n u m b e r o f factors are anticipated to influence the individual h e m e redox potentials. F i r s t a n d foremost, the environment o f each heme c a n exert an influence o n its o x i d a t i o n - r e d u c t i o n potential. T h i s influence w i l l b e manifested i n two ways: the p a c k i n g o f the specific amino a c i d side chains about each h e m e a n d the extent o f solvent exposure o f each h e m e . It is quite apparent f r o m the structural data (Figures 1 a n d 2) that the four hemes, w h i c h are i n nonequivalent environments, are expected to have different o x i d a t i o n - r e d u c t i o n poten tials. M o r e o v e r , at least w i t h M i y a z a k i cytochrome c , one o f the hemes (heme II) is substantially m o r e exposed to solvent, w h i c h m a y result i n a lower o x i d a t i o n - r e d u c t i o n potential (13). F i n a l l y , it is apparent that i n a small molecule that contains four hemes w i t h i n close p r o x i m i t y ( < 18 A ) , h e m e - h e m e interactions, principally as a result o f electrostatic interactions, are likely to influence o x i d a t i o n - r e d u c t i o n potentials (14). Indeed, o n elec trostatic grounds the redox state o f one heme should influence another. T h i s influence results f r o m the fact that addition o f electrons changes the f o r m a l 3

3


478


charge due to each h e m e iron f r o m plus to zero, so the electrostatic experienced b y adjacent hemes w i l l b e significantly altered.

field

In biological redox proteins containing t w o o r m o r e redox centers two types o f o x i d a t i o n - r e d u c t i o n potentials c a n b e defined. T h e f o r m a l potential o f the molecule w i l l result i n a macroscopic redox potential for each o f the four hemes, w h i c h reflects the fact that five macroscopic states exist: S , fully oxidized; S one electron r e d u c e d ; S , t w o electron reduced; S , three electron reduced; a n d S , fully r e d u c e d ( F i g u r e 3) (14). H o w e v e r , each individual h e m e has a definable o x i d a t i o n - r e d u c t i o n potential (the microscopic potential), w h i c h m a y vary d e p e n d i n g o n the redox state o f its neighbors. A s shown i n F i g u r e 3 f o r cytochrome c , a total o f 32 microscopic potentials may exist. These potentials c a n b e thought o f as descriptions o f the distribution o f electron density as electrons partition a m o n g the four hemes. F r o m the microscopic potential, an interaction potential between any two hemes (heme i a n d h e m e j) c a n b e d e f i n e d as the change i n the m i d p o i n t redox potential o f h e m e i caused b y the oxidation o f h e m e j. F o r example, the interaction potential between the hemes w i t h the t w o highest macro scopic potentials, h e m e 1 a n d h e m e 2, is the difference between the redox 0


1 ?

2

3

4

3

SQ

S2

S4

S3

Figure 3. Electron distribution for cytochrome c . S -S define the five macroscopic states (see text). The 16 microscopic states are shown. Open circles represent oxidized heme, and solid circles represent reduced heme. The microscopic redox potentials of heme i are given by e^ where j , k, 1 represent hemes that remain oxidized. For clarity only, 12 of the 32 microscopic redox potentials are labeled. 3

0

4

kl


CUSANOVC IH ET AL. Cytochrome

22.

potential o f heme 1

c : Properties and Recent Insights

479

3

a n d the redox potential o f h e m e 1 w h e n h e m e 2 is

oxidized (ef ), w h i c h is t e r m e d i

1

2

( 1 5 ) . T h e nomenclature u s e d here is f r o m

Santos et a l . ( 1 5 ) a n d is illustrated i n F i g u r e 3. T h e h e m e notation does not relate to structural assignments (Figures 1 a n d 2) b u t to redox potential only. Table II summarizes the macroscopic redox potentials for the four cytochromes c . A s expected f r o m the strong amino acid sequence homology, 3

the M i y a z a k i a n d H i l d e n b o r o u g h cytochromes c

have redox potentials that


3

are very similar a n d have a redox potential span o f 1 1 0 - 1 2 0 m V between heme 1 a n d h e m e 4. I n sharp contrast, N o r w a y cytochrome c

3

has a redox

potential span o f approximately 235 m V . Clearly, the differences i n macro scopic redox potentials a m o n g the various cytochromes c

3

result f r o m the

different amino acid sequences a n d , thus, different h e m e environments. T o date, little can b e said about the specific reasons for differences i n macro scopic redox potentials a m o n g the cytochromes c , b u t i n v i e w o f the large 3

amount o f structural information that is accumulating, m u c h progress c a n b e expected i n the future. I n terms o f the microscopic potentials, the interaction potentials are o f particular interest because they provide i n f o r m a t i o n o n the interaction o f the individual hemes w i t h each other (15-19). T a b l e I I I presents interaction

Table IL Cytochrome c Macroscopic Oxidation-Reduction Potentials 3

E ' (mV)

a

0

Source Miyazaki Hildenborough Norway Gigas

1

2

3

4

Reference

-260 -263 -165 -235

-312 -321 -305 -235

-327 -329 -365 -306

-369 -381 -400 -315

14 16 17 18

Potentials referred to the normal hydrogen electrode at p H values near 7.

a

Table III. Interaction Potentials from Microscopic Redox Potentials Interaction Potentials (mV) hz hs *14

^23 ^24 Î34 a

Miyazaki Cytochrome pH7.1 5 -21 -35 43 -11 -7

c

a

3

Gigas Cytochrome pH7.2

c

3

19 -26 6 42 -24 -18

Reference 19. Reference 15.


480


potentials for the M i y a z a k i a n d Gigas cytochromes. G i v e n the very different macroscopic potentials, the similarities between the Gigas a n d M i y a z a k i interaction potentials are striking. O n l y I shows a different sign, a n d the actual values o f the other interaction potentials are quite similar given the precision o f these difficult measurements.


l4

E l e c t r o c h e m i s t r y cannot provide direct i n f o r m a t i o n about the individual hemes and, thus, a spectroscopic technique is n e e d e d . I n m o n i t o r i n g the redox state o f each h e m e d u r i n g titration to p r o d u c e diamagnetic F e ( I I ) , it is possible to take advantage o f the paramagnetic properties o f the F e ( I I I ) h e m e core using N M R spectroscopy (15, 19, 20). T h i s methodology has also b e e n used to establish a l o w e r b o u n d a r y for the rate constant for intramolecu lar electron transfer i n cytochrome c . W i t h Gigas the intramolecular elec tron-transfer rate constant must be greater than 1 0 s ~ (15), a n d recent w o r k suggests a rate constant o f approximately 1 0 s for M i y a z a k i cy tochrome c ( K . K i m u r a , S. Nakajima, a n d K . N i l d , personal communication). T h e rate o f intermolecular electron transfer for M i y a z a k i cytochrome c is m u c h lower, w h i c h makes it possible to distinguish intramolecular f r o m intermolecular processes (19). T h i s distinction w i l l prove to be a valuable property for fundamental studies. M o r e o v e r , using N M R , F a n et al. (14) c o n c l u d e d that h e m e 1 (the highest potential h e m e ) i n the redox studies can be related to h e m e I i n the crystal structure, a n d that H e m e 3 i n the macroscopic redox titrations is h e m e I V i n the structure. T h u s , w e w i l l adopt the nomenclature h e m e 1(1) a n d h e m e IV(3) to relate macroscopic redox potential to structure. 3

5

8

1

- 1

3

3

A l t h o u g h complex, the ability to assign microscopic redox potentials a n d to determine interaction potentials sets the stage for the analysis o f the redox data i n terms o f the structure o f cytochrome c . T h i s approach s h o u l d greatly expand o u r understanding o f biological redox potentials a n d facilitate efforts to exploit a n d understand the electrochemical, electrical, a n d kinetic proper ties o f cytochrome c . 3

3

Heterogeneous Electron Transfer A striking feature o f the cytochromes c is their ability to m o d i f y an electrode surface i n such a way as to facilitate subsequent electron transfer (21). A m o n g the different cytochromes studied to date, only cytochrome c can be oxidized a n d r e d u c e d w i t h h i g h electrochemical reversibility at an e l e c t r o d e - s o l u t i o n interface w i t h o u t any mediator or p r o m o t e r (16, 21). T h i s reversibility contrasts w i t h the m o r e usual situation i n w h i c h the p r o t e i n absorbs o n the electrode surface, thus preventing further electron transfer. Alternatively, rather slow (k° < 1 0 " c m / s ) heterogeneous electron transfer rates are observed. U s i n g M i y a z a k i cytochrome c a n d a m e r c u r y electrode on w h i c h specific adsorption o f cytochrome c is k n o w n to occur, the ad3

3

3

3

3


22.

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481

3


sorbed film o f the p r o t e i n remains electrochemically active a n d continues to facilitate heterogeneous electron transfer between the electrode a n d the soluble p r o t e i n . Because the heterogeneous electron-transfer rate was extremely fast, it was not possible to evaluate it b y conventional techniques such as cyclic voltammetry. Instead, the galvanostatic double pulse m e t h o d was e m p l o y e d (21). F r o m these measurements, a heterogeneous rate constant o f 1.2 c m / s ( a = 0.48) was obtained. T o o u r knowledge, this is the first example o f a directly measured p r o t e i n heterogeneous rate constant o f such a large magni tude. D i f f e r e n t i a l capacitance measurements suggest that monolayer cover age o f cytochrome c is achieved a n d that electron transfer occurs t h r o u g h the adsorbed p r o t e i n layer that possesses essentially " n a t i v e " properties. T h e electrode is therefore " m o d i f i e d " b y the electroactive species itself. T o consider w h e t h e r this k i n d o f m o d e l is reasonable, it is necessary to estimate the rate o f electron transfer through the film. F r o m N M R measurements, the intra- a n d intermolecular electron-transfer rate constants can b e estimated. These values are fc = 1.5-7.8 Χ 10 s " a n d fe = 1 Χ 10 M /s, respectively ( K . K i m u r a , S. Nakajima, a n d K . N i k i , personal communication). T h e u p p e r l i m i t for the heterogeneous rate constant c a n b e estimated f r o m M a r c u s theory (eq 2): 3

8

intra

1

inter

_ 1

4

fc°^Z (fc /Z ) hetero

ex

ex

(2)

1/2

where Z and Z are the collision frequencies o f a molecule w i t h an electrode (heterogeneous) a n d i n solution (homogeneous), respectively (22). I n this case k = k , a n d the estimation concludes that k° < 3 c m / s . T h e steric a n d molecular association terms are assumed to b e unity. h e t e r o

e x

ex

inter

A n electron-transfer reorganization energy, X , o f 0.94 e V (21) was also calculated f r o m M a r c u s theory (22). T h i s value is very close to that o f the F e ( I I I ) - F e ( I I ) system i n acidic m e d i u m (23) a n d to cytochrome c (0.7 e V ) a n d cytochrome b (1.2 e V ) f r o m measurements o f self-exchange (24). A s far as w e are aware, this is the first example o f a consistent X value for a h e m e protein measured electrochemically a n d b y self-exchange measurements. 5

Recently it was p r o p o s e d that the apparently slow heterogeneous elec tron-transfer rates for such proteins as cytochrome c, cytochrome b , plastocyanin, a n d ferredoxin are an artifact o f the experimental approach (25). Instead o f assuming that p r o t e i n molecules react at a planar a n d essentially homogeneous surface, it is assumed instead that movement o f the p r o t e i n occurs predominantly b y radial diffusion to very small, specific sites. These sites are p r e s u m e d to facilitate very r a p i d electron transfer at the reversible potential w h i l e the rest o f the surface remains inactive. T h u s , the m o d i f i e d electrode surface behaves like an array o f microelectrodes. I f this theory is used to treat previous data, m u c h higher electron-transfer rate constants are obtained. A l t h o u g h this theory deserves m o r e detailed scrutiny, it m a y serve 5


482


as a contrast to the case o f cytochrome c w h e r e the entire surface is " a c t i v e " and, therefore, classical models o f macroscopic planar or spherical diffusion can be applied. 3

A d s o r b e d layers o f cytochrome c have also b e e n u s e d to facilitate electron transfer o f molecules that otherwise w o u l d not y i e l d h i g h electrontransfer rates. F a c i l i t a t e d electron transfer has b e e n observed for flavodoxin a n d ferredoxin o n cytochrome c m o d i f i e d basal plane pyrolytic graphite (26). 3


3

Surface Spectroscopy Because cytochrome c films have u n i q u e properties, a n effort has b e e n made to study such deposits b y spectroscopic techniques, especially surfaceenhanced R a m a n spectroscopy ( S E R S ) . W h e n M i y a z a k i cytochrome c is adsorbed o n a suitably treated silver electrode, S E R S spectra may be ob tained. M a r k e r bands at 1380 a n d 1375 c m have b e e n u s e d to characterize the redox state o f the adsorbed p r o t e i n (27). I f the potential a p p l i e d to the electrode is varied, an apparent redox potential for the adsorbed film can be measured. A value o f —260 m V versus the n o r m a l hydrogen electrode ( N H E ) was obtained. T h i s value corresponds to the highest o f the four macroscopic redox potentials for M i y a z a k i cytochrome c ( — 260 m V ) . B e cause the S E R S effect is most p r o n o u n c e d for the interactions o f the p r o t e i n w i t h the surface over distances o f about 5 A , it may be assumed that the observed interaction is d o m i n a t e d b y the h e m e closest to the surface. T h i s conclusion is consistent w i t h the environment a r o u n d this heme, w h i c h is least " e x p o s e d " a n d is also i n a h i g h lysine e n v i r o n m e n t — c o n d i t i o n s likely to p r o m o t e c h e m i s o r p t i o n a n d interaction because the electrode is operated o n the negative side o f the point o f zero charge. Based o n the structural information available, this w o u l d suggest that cytochrome c is oriented o n the surface a n d h e m e I ( F i g u r e 1) is the closest to the surface. I n contrast, i f silver c o l l o i d is deposited o n a M i y a z a k i cytochrome c film adsorbed o n a silver electrode, the S E R S signal is d o m i n a t e d b y the c o l l o i d interaction a n d a m u c h lower ( — 300 m V versus N H E ) potential is obtained. T h i s l o w e r potential w o u l d fall i n the m i d d l e o f the range o f macroscopic potentials for M i y a z a k i cytochrome c . It is, therefore, likely that the c o l l o i d measures all h e m e sides, whereas the silver electrode itself reveals more specific interac 3

3

- 1

3

3

3

3

tions. T h e foregoing arguments assume that interaction o f the silver w i t h the protein does not p e r t u r b the redox potentials, an assumption that may not be valid. T h e resonance R a m a n spectrum o f H i l d e n b o r o u g h cytochrome c adsorbed o n a highly oriented pyrolytic graphite electrode is very weak. H o w e v e r , w h e n silver c o l l o i d is deposited o n the adsorbed protein, a signal nearly as large as that previously n o t e d is observed. T h o u g h these results are preliminary, they indicate the possibility o f d e t e r m i n i n g apparent redox 3


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potentials b y following the intensity o f marker bands as a potential is a p p l i e d to a substrate (electrode surface) that is essentially S E R S - i n a c t i v e . T h e use o f S E R S to study the properties o f adsorbed films o f cytochrome c is b e i n g p u r s u e d at the present t i m e . 3

It s h o u l d b e n o t e d that resonance R a m a n studies o f cytochrome c show evidence o f b a n d spfittings i n partially r e d u c e d solutions (28). T h i s observa tion suggests either nonequivalence o f the four hemes o r an exciton-like splitting o f vibrational modes. H e m e - h e m e interactions are clearly important.


3

Electrical Properties of Protein Films F i l m s o f cytochrome c show m o r e than 10 orders o f magnitude change i n resistivity ( 1 0 - 5 7 Ω - c m ) w h e n they pass f r o m the fully oxidized to the fully r e d u c e d state ( 2 9 ) . T h e r e have b e e n a n u m b e r o f studies o f electrical conductivity o f proteins a n d p r o t e i n films. T h i s conductivity c a n result f r o m electric-field-induced diffusion o f charge carriers: cations a n d anions, p r o tons, electrons, a n d holes. I n the case o f cytochrome c , the large difference i n resistivity can be d u e to a change o r changes i n polarization energy, changes i n electronic states o f h e m e clusters, a n d interactions between hemes. T h e presence o f absorbed water, w h i c h can comprise a significant part o f the protein v o l u m e , can have a strong influence o n the electrical properties. Rosenberg (30) measured the conductivity o f h e m o g l o b i n as a function o f absorbed water a n d explained the increase i n conductivity as a result o f a change i n dielectric constant coincident w i t h absorption o f water. H o w e v e r , this explanation is controversial. E l e y (31) has suggested that conduction i n " w e t " p r o t e i n is i o n i c . T a k a s h i m a a n d Schwan ( 3 2 ) , w h o studied crystalline powders o f proteins, f o u n d that the increase i n dielectric constant is proportional to the increase i n adsorbed water u n t i l the first adsorption layer is c o m p l e t e d . A b o v e two o r three layers, the dielectric constant does not increase further. Presently, it is assumed (30) that b e l o w 1 5 % adsorbed water, the conductivity o f a p r o t e i n is almost entirely electrical i n nature. E l e c t r o n i c c o n d u c t i o n can b e intrinsic or extrinsic a n d can b e p r o m o t e d b y charge transfer o r b y t u n n e l i n g (33). Previous studies suggested that some hydrated proteins can behave like semiconductors. A s a semicon ductor, a p r o t e i n c a n be expected to show increasing conductivity w i t h increasing temperature. O x i d i z e d cytochrome c shows exactly the opposite 3

1 2

3

3

effect, w h i c h suggests behavior like an ionic conductor. H o w e v e r , r e d u c e d cytochrome c i n d r y films does show an increase i n conductivity w i t h an increase i n temperature that is consistent w i t h a semiconductor (34). Some particular changes o f resistivity o f cytochrome c w i t h temperature have b e e n observed. B e l o w the temperature o f 292 Κ a n d u n d e r 200-kPa hydrogen pressure, ferrocytochrome c has a n activation energy o f 2.15 e V , w h i c h reflects the semiconductor nature o f the p r o t e i n . A b o v e this temperature, 3

3

3


484


b o t h f e r r i - a n d ferrocytochrome forms are b e l i e v e d to exist (34). A b o v e a critical temperature o f 346 K , the activation energy drops to 1.55 e V . This drop causes the difference i n resistivity between the oxidized a n d r e d u c e d forms. A t the present time, the behavior o f cytochrome c i n dry films is not w e l l understood i n molecular terms. H o w e v e r , an i n - d e p t h understanding o f the electrical properties o f cytochrome c may be most useful i n p r e p a r i n g biological materials for applications i n bioelectronics. 3


3

Homogeneous Electron Transfer T h e kinetics o f oxidation a n d reduction o f the cytochromes c provide a means to better understand their physicochemical a n d functional properties. H o w e v e r , because o f the complexity o f m u l t i p l e hemes, l o w redox potentials, and sensitivity to molecular oxygen, kinetic studies have b e e n l i m i t e d c o m p a r e d w i t h other c-type cytochromes a n d redox proteins. Nevertheless, some information is available that provides insight into the mechanism o f action o f cytochrome c . 3

3

T h e reduction o f b o t h H i l d e n b o r o u g h a n d N o r w a y cytochrome c b y S 0 ( S 0 * ~ ) is biphasic, a n d b o t h phases are second order ( S 0 * is the reactive species) (35, 36). A t p H 9.1 the two kinetic phases are approximately equal i n magnitude, w h i c h suggests that the four hemes can be d i v i d e d into two groups o f two, w i t h rate constants o f 6.8 Χ 1 0 a n d 2.1 Χ 1 0 M /s, respectively (35). A t p H 7.0 the fast phase accounts for 8 6 % o f the reaction w i t h N o r w a y cytochrome c (k = 1 Χ 1 0 M / s ) a n d 7 2 % w i t h H i l d e n b o r ough cytochrome c (k = 3.2 Χ 1 0 M / s ) ( 3 6 ) . T h e slow phase was f o u n d to be 10- to 2 0 - f o l d smaller than the fast kinetic phase at p H 7. T h e p r o b l e m is that the biphasic kinetics suggest a slow intramolecular electron transfer. T h i s result is i n conflict w i t h the N M R data that indicate fast intramolecular electron transfer (15, 19). D i t h i o n i t e is notoriously difficult to use a n d causes a variety o f difficulties. I n v i e w o f foregoing difficulties, the N M R results, a n d (as w i l l be shown) the very different results w i t h other reactants ( i n c l u d i n g the physiological reactant ferredoxin), it is probably best to v i e w the d i t h i o n ite results as an artifact. U s i n g the methylviologen cation radical ( M V * ) f o r m e d b y pulse radiolysis, monophasic kinetics o f cytochrome r e d u c t i o n are observed w i t h a rate constant o f 4.5 Χ 1 0 M / s (1.1 Χ 1 0 M / s o n a p e r h e m e basis) at p H 8.0 w i t h the H i l d e n b o r o u g h cytochrome (36). T h i s very fast second-order process approaches the diffusion controlled limit. M o r e o v e r , the reverse reaction can be estimated to be 7.8 Χ 1 0 M / s , w h i c h suggests that the reaction takes place p r i m a r i l y w i t h the highest potential heme (the AE' between h e m e I and M V is 190 m V , consistent w i t h an e q u i l i b r i u m constant of approximately 1 0 ) . Interestingly, the kinetics w i t h M V * are ionic strength dependent, w h i c h is consistent w i t h a p l u s - p l u s interaction, 3

2

4

2

2

2

6

7

3

6

3

6

_ 1

- 1

_ 1

+

8

_ 1

8

_ 1

4

_ 1

0

# +

3


+

22.

CUSANOVICH E T AL.


485

3

and suggest a net charge o f w h i c h is a result consistent M o r e o v e r , the M V * results transfer, as p r e d i c t e d w i t h Ν +

+ 4.7 + 0.7 o n H i l d e n b o r o u g h cytochrome c , w i t h that expected f r o m the isoelectric point. are consistent w i t h fast intramolecular electron MR. 3

Recently w e carried out kinetic studies w i t h H i l d e n b o r o u g h a n d M i y a z a k i cytochrome c using deazariboflavin semiquinone ( d R f * ) , M V * , a n d propy lene diquat ( P D Q ) , p r o d u c e d b y laser flash photolysis, as reductants ( 3 7 ) . Initially, a l l three reactions were accurately second order, consistent w i t h a l l hemes b e i n g r e d u c e d w i t h the same rate constant o r w i t h a single site reduced, f o l l o w e d b y fast intramolecular electron transfer to reduce the r e m a i n i n g three hemes. H o w e v e r , b y measuring r e d u c t i o n kinetics w i t h cytochrome c poised at different extents o f reduction, the kinetics o f reduction o f individual hemes c o u l d b e resolved. T h u s , reduction o f cy tochrome c i n approximately 5 % steps a n d application o f the k n o w n macroscopic redox potentials (see previous section) enabled calculation o f the concentration o f each h e m e (c^ at each stage o f reduction. T h e plot o f k versus percent reduction can thereby be fitted to solve f o r the rate constant for each h e m e (k^: +

3


e +

3

3

ohs

fc

obs

= LcA

(3)

U s i n g the macroscopic potentials o f H i l d e n b o r o u g h cytochrome c at l o w ionic strength (16 m M ) , the rate constant for reduction o f the h i g h potential heme b y d R P is 1 Χ 1 0 M / s a n d f o r the other three hemes is 4.2 Χ 1 0 M / - Similarly, for M i y a z a k i cytochrome c rate constants o f 8 X 1 0 a n d 5.2 Χ 1 0 M ~ V s were obtained. W i t h P D Q , the lowest a n d highest potential hemes have the same kinetics (k = k = 1.6 Χ 1 0 M / s ) i are resolvable f r o m the t w o intermediate potential hemes (k = k = 0.6 X 1 0 M / s ) f o r H i l d e n b o r o u g h cytochrome c . M o r e o v e r , the reduction kinetics w i t h M V * a n d P D Q * are ionic strength dependent a n d the rate constant increases w i t h increasing ionic strength as expected f o r a p l u s - p l u s electrostatic interaction. H o w e v e r , w e find that the r e d u c t i o n kinetics w i t h d R f * are also ionic strength dependent, w h i c h is unexpected because d R f * is uncharged u n d e r the experimental conditions used. T h i s unexpected result establishes that electrostatic analysis o f the cytochromes c is c o m p l i c a t e d b y an effect o f ionic strength o n the redox potential o f one o r more hemes o r an ionic-strength-induced structural change. Nevertheless, results to date estab lish that b y controlling the driving force (i.e., redox potential o f the reductant), the electrostatics (charge o n the reductant as w e l l as ionic strength), a n d the extent o f r e d u c t i o n o f cytochrome c , the interaction domains o f i n d i v i d u a l hemes can be p r o b e d . T h i s type o f approach has b e e n a p p l i e d to a w i d e variety o f redox proteins to y i e l d i n f o r m a t i o n o n the topology a n d electrostat ics o f their molecular surface (38, 3 9 ) . Clearly, the cytochromes c are m o r e 3

9

_ 1

_ 1

8

s

8

3

8

# +

]

8

4

- 1

2

8

_ 1

3

3

+

+

3

3

3


a

n

(

486


complex than systems containing a single prosthetic group, b u t they are amenable to analysis. Based o n o u r kinetic analysis a n d the finding that the lowest potential h e m e is either as reactive as the intermediate potential hemes ( d R f * ) o r more reactive ( P D Q * ) , w e c a n conclude that the l o w potential h e m e is the most exposed h e m e (heme II, F i g u r e 2). T h i s assignment is supported b y the fact that solvent exposure tends to stabilize the oxidized f o r m o f hemes relative to the r e d u c e d f o r m a n d results i n a lower m i d p o i n t potential ( 1 3 ) . T h u s , c o m p l e t i n g the relation o f redox potential to structure [heme 1(1) a n d heme IV(3)], w e c a n make additional assignments: h e m e 11(4) a n d , b y deduction, h e m e 111(2). T h i s relationship between h e m e macroscopic redox potential a n d structure s h o u l d greatly facilitate future kinetic studies that relate structure to function. T h e s e assignments differ f r o m those r e p o r t e d for the N o r w a y cytochrome based u p o n E P R measurements (40), w h e r e the highest potential h e m e is h e m e I V , f o l l o w e d b y h e m e 1(2) a n d h e m e 111(3). T h e only consistency between the N o r w a y a n d M i y a z a k i h e m e - r e d o x poten tial assignments is f o r h e m e II, w h i c h has the lowest redox potential. A l t h o u g h the specific reasons f o r these differences are not yet understood, the apparent lack o f agreement is not surprising i n light o f the l o w sequence homology.


+

C y t o c h r o m e c can f o r m stable complexes w i t h its physiological reactant ferredoxin (dissociation constant o n the o r d e r o f 1 μ Μ ) (41). T h e reactions o f oxidized ferredoxin w i t h r e d u c e d cytochrome c a n d r e d u c e d ferredoxin w i t h oxidized cytochrome c are r a p i d ( > 1 0 M / s ) ( 3 6 ) . M o r e o v e r , electron transfer w i t h i n the f e r r e d o x i n - c y t o c h r o m e c complex c a n b e measured a n d is approximately 150 s ( 3 6 ) . Recently, hypothetical complexes between cytochrome c a n d ferredoxin w e r e p r e p a r e d using molecular graphics (41). C o m p l e m e n t a r y charge interactions w e r e f o u n d that are consistent w i t h relatively strong b i n d i n g constants at l o w ionic strength. M o r e o v e r , n o severe steric restrictions are f o u n d that prevent relatively close approach o f pros thetic groups. A l t h o u g h not definitive, computer-generated models provide a basis f o r designing experiments to elucidate the interactions m e d i a t i n g c o m plex formation. 3

3

7

_ 1

3

- 1

3

T h e kinetic studies c o m p l e t e d to date support the n o t i o n o f r a p i d intramolecular electron transfer. M o s t importantly, kinetic studies establish that u n d e r appropriate conditions the kinetics o f individual hemes c a n b e p r o b e d . H o w e v e r , to exploit fully the cytochrome c system, w e n e e d a 3

detailed understanding o f the perturbations caused b y changing the ionic strength. T h i s area w i l l b e a major focus o f future studies.

Site-Directed Mutagenesis A n important vehicle to exploit a n d understand the u n i q u e properties o f the cytochromes c is site-directed mutagenesis. T h i s approach allows perturba3


22.


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3

tion o f specific features o f cytochrome c . I n c o m b i n a t i o n w i t h structural studies (X-ray a n d N M R ) , redox titrations, kinetic properties, a n d electrical properties, site-directed mutagenesis c a n b e e m p l o y e d as a p o w e r f u l t o o l f o r elucidating the role o f specific amino a c i d side chains. T o this e n d , w e recently c l o n e d a n d expressed cytochrome c f r o m H i l d e n b o r o u g h (42). T h e H i l d e n b o r o u g h gene was isolated some time ago (43), b u t Desulfovibrio is not a convenient host f o r site-directed mutagenesis. H i l d e n b o r o u g h cy tochrome c can b e expressed i n E. colt, b u t only the apoprotein is p r o d u c e d (44). W e have n o w expressed H i l d e n b o r o u g h cytochrome c i n Rhodobacter sphaeroides, w h e r e it is p r o p e r l y processed a n d has properties identical w i t h wild-type cytochrome c (42). 3


3

3

3

3

Mutants are currently b e i n g p r e p a r e d to alter the properties o f cy tochrome c . T o illustrate this approach, several examples c a n be described: replacement o f his-70 b y m e t a n d ala, substitution o f lys-15 w i t h asp, a n d mutation o f phe-20 to l e u . Replacement o f his-70 w i l l alter the ligation o f h e m e 1(1) a n d force h i g h spin (ala substitution) o r h i g h potential (met substitution). These changes should substantially alter the redox potential o f h e m e 1(1) a n d thus affect kinetic a n d electrical properties. Similarly, replace ment o f lys-15 b y asp w i l l alter the positive electrostatic field i n the vicinity o f h e m e 1(1). T h i s mutation should affect the ability o f cytochrome c to interact o n metal surfaces a n d may alter its properties i n dry films. Phe-20 is conserved i n the amino a c i d sequence o f a l l tetraheme Desulfovibrio cy tochromes c a n d is positioned between the two intermediate redox potential hemes, heme 111(2) a n d h e m e IV(3). T h u s , this mutation may have p r o f o u n d effects o n intramolecular electron transfer i n cytochrome c . Obviously, a large n u m b e r o f mutations c a n b e p r e p a r e d that m a y play critical roles i n understanding the cytochromes c a n d addressing the questions p o s e d here. 3

3

3

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3

Summary A l t h o u g h complex, the cytochromes c provide the opportunity to obtain information that w i l l greatly extend o u r knowledge o f biological electron transfer a n d the interaction o f redox centers i n m u l t i h e m e proteins. M o r e over, because o f u n i q u e electrochemistry a n d electrical properties, the cy tochromes c provide the opportunity to develop a system useful as a m o d e l for bioelectronic devices. M u c h research remains to b e done to understand fully the redox properties o f the cytochromes c . H o w e v e r , the data discussed clearly define interesting a n d important issues, w h i c h i n c l u d e (1) the paths b y w h i c h electrons move between hemes; (2) h o w electrons enter a n d exit the cytochrome c molecule d u r i n g physiological electron transfer; (3) the nature o f the factors that c o n t r o l the interaction potentials between hemes; (4) the factors responsible f o r the observed behavior o n metal surfaces; and, i m p o r tantly, (5) the specific molecular features responsible f o r the behavior o f 3

3

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488

BIOMEMBRANE

ELECTROCHEMISTRY

cytochrome c i n d r y films. F r o m the structural data discussed here, a n intuitive m o d e l c a n b e described that may explain some o f the properties o f the cytochromes c . G i v e n the asymmetric charge distribution o n the cy tochrome c surface ( F i g u r e 2) a n d the close proximity o f the metal centers w i t h i n the molecule, it is plausible that the molecule c a n orient o n metal surfaces o r i n films i n a n ensemble o f p l u s - m i n u s interactions that create what c o u l d b e v i e w e d as a n oriented h e m e ( F e ) w i r e . A l t h o u g h m u c h more w o r k must b e done to prove this concept, it does provide a basis f o r experimental design. 3

3


3

Acknowledgment T h i s w o r k was supported b y a grant f r o m the Office o f N a v a l Research.

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RECEIVED for review January 29, 1992.

1991.

ACCEPTED revised manuscript


July 22,

Properties and Recent Insights - American Chemical Society

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