Comparative studies of monohemic bacterial C-type cytochromes. Redox and optical properties of Desulfovibrio desulfuricans Norway cytochrome c553(550) and Pseudomonas aeruginosa cytochrome c551

Vol. 113, No. 2, 1983 June

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 526-530

15, 1983

COMPARATIVE STUDIES OF MONOHEMIC BACTERIAL

C-TYPE CYTOCHROMES.

REDOX AND OPTICAL PROPERTIES OF DESULFOVIBRIO CYTOCHROME C553(55D) Pierre

Jean HALADJIAN", and Mireille

de Chimie de Provence,

**Laboratoire

Received

April

NORWAY

AND _PSEUDOMONAS AERUGINOSA CYTOCHROME C55I

BIANCO",

'Laboratoire Universite

DESULFURICANS

Mohammed LOUTFI**

BRUSCHI%*

et Electrochimie 13331 Marseille

des Complexes, Cedex 3 (France)

de Chimie Bacterienne, C.N.R.S., 13277 Marseille Cedex 9 (France)

B.P.

71,

5, 1983

from Desulfovibrio desulfuriRedox properties of cytochrome c553(550 cans Norway (EA = 0.04 + 0.02 V/NHE) and cytoc II rome ~551 from P aeruginosa TE;;-= 0.25 t 0.02 V/NHEr are compared with those of some monoh&ic c-type cytochromes. The pK value for the equilibrium between the pH-dependent forms of cyIt is to be notochrome ~553 550) (pK = 10.3 + 0.1) has been also determined. ted that the 6 ifference between redox potentials can extend to nearly 250 mV, though the axial heme ligands are identical. Structural reasons have to be invoked to explain these varlations.

C-type

cytochromes

tems

(1).

zed,

and the properties

ligands

Several

mono-

and by the

dox potential, structure

constitutes

(3)

we have obtained

hemic

cytochrome

vulgaris

Hildenborough

in agreement the

iron

raised,

are It

correlating

sys-

been characteriby the iron

One of these

for

when comparing

known

that

V/NHE (2)).

a redox

potential

isolated

from

extracted

properties,

redox

a transition

re-

behavior,

c has a

in a recent

bacteria.

monohemic

iron-methionine

526

cytochrome hand,

of work

-t 0.01 V/NHE for the monomicroorganism Desulfovibrio

reducing

In these

$1.50

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

potentials

of 0.02

and histidine

between

redox

mitochondrial

anaerobic

sulfate

(4).

to methionine

the

On the other

value the

from

EPR measurements linked

have

seem to be controlled

basis

observed

is well

(0.255

cytochromes

transport

hemoproteins.

can be observed.

0006-291X/83 Copyright A/l rights

with

atom is

when pH is forms

c553

group

of electron

environment.

a powerful

c.

potential

c-type

proteic

of these

features

cytochromes

positive

in a variety

of the hemic

and functions

monohemic

present

and polyhemic

immediate

Intriguing very

are

This

c-type

residues linked

value

cytochromes,

at neutral

pH ;

and unlinked

is

Vol. 113, No. 2, 1983

BIOCHEMICAL

In the present hemic

bacterial

reducing

work,

the

cytochromes

bacteria

Norway)

and the

aeruginosa).

other

comparison

c

one from

from

from

553(550) an aerobic

The purification

tive

cytochrome

and depends In the

to study

bacteria

to other species

desulfuricans

(cytochrome

~55~ from

and the physicochemical

it

presents works

seems that

mono-

of sulfate

properties

a "split (5,6).

Pseuof

cl" band in the

In the case of

the redox

potential

is

very

posi-

on pH (7).

present

redox

c551 with

~5~~'

another

Desulfovibrio

cytochrome c553(550) which -D. desulfuricans reduced form have been reported in previous P. aeruginosa

RESEARCH COMMUNICATIONS

has been extended

c : one isolated

(cytochrome

domonas

AND BIOPHYSICAL

work,

voltammetry

and optical

and spectrophotometry

properties

the aim of comparing

of cytochrome

have been used

c

and cytochrome 553(550) of monohemic c-type cytochromes.

some properties

EXPERIMENTAL Materials. Cytochrome c553(550) from D. desulfuricans chrome ~551 from P. aeruginosa were prepared and purified bed (6,8). All chemicals used were reagent grade. Methods and apparatus. The working surements was the 4 4'-bioyridine activated details are given i; a previous work (3).

Norway and cytoas previously descri-

electrode used for voltammetric meagold electrode ; all experimental

RESULTS Redox properties. chrome c553(550) bipyridine (curve dic

and anodic

voltammetry after

l),

cyclic

In the presence

The differential

solution

conversion

voltammograms

in Fig.

la.

voltammetry

in Fig.

peaks

M 4,4'-bipyridine, in Fig.

lb (curve

a well-shaped

at E

= - 0.15 P la (curve 2).

separation

Likewise,

catho-

very

slight. pulse

0.03

+ 0.01

cathodic

V/NHE

and anodic

(after correction in Fig. lb (curve

for 2).

backThe

= E - Epc % 60 mV is consistent with the theoretical value (9) pa diffusion-controlled monoelectronic process. Moreover, peaks currents ipa and i were found to increase linearly with v 112 (v = scan rate) PC as expected for a diffusion controlled process (9). The midpoint potential for

AE

of 4,4'-

1) are

(i.e.

Well-developed

at E = - 0.19 V and E = - 0.13 V/Ag-AgCl PC pa ground current) are detected by cyclic voltammetry

absence

differential

V/Ag-A$1

peaks

of a 79 r.lM cyto-

In the

peak can be detected.

observed

(3))

pulse

are shown

no well-developed

of 0.01

peak is

pH 7.0

P

a reversible

between

E

and E

potentialPT1O) an average cyclic

, which

isPCfound value

voltammetry

of 0.04

for

a reversible

at - 0.16

-+ 0.02 measurements

V/An

- AgCl

V/NHE resulting is

process

estimated

corresponds

(i.e. 0.05 -t 0.02 -from differential for

the

redox

c553(550)' Similar curves have been obtained for -P. aeruginosa a value of Eo = 0.25 + 0.02 V/NHE has been determined, in vious results (7). -

to the

redox

V/NHE).

Thus,

pulse

potential

and

of cyto-

chrome

527

cytochrome agreement

~55~ with

;

pre-

Vol. 113, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

W

(0) i

Effect of 4,4/-bipyridine on the electrochemical reactions at the g iold electrode of 79 uM cytochrome c553(550) in 0.05 M sodium phosphate buffer at pH 7.0 : (a) differential pulse voltammograms, scan rate 2 mV s-l (b) cyclic voltanxnograms, scan rate 1 mV s-l (1) in the absence (2 in the presence of 0.01 M 4,b-bipyridine ; (....) background solutions.

Effect form

of pH. The spectrum

shows an absorption

thionine

ligand

in Fig.

2 (curve

pH 'L 9.5 band

the

absorbance

decrease

From the

("neutral")

is

for

the

of the state

the

oxidized

interaction

raised,

of the 690 nm band noted

("alkaline")

form

in

to the

When pH is

disappearance

a new conformational

the native

(1,ll).

heme iron

1) that

2) (3).

c553(550)

band at 690 nm attributed the

; a simultaneous

(curve

med that

with

of cytochrome

it

observed

decreases

absorbance

above

of the

690 nm band, appears,

of meis

it

529 nm

may be assu-

in equilibrium

with

as follows

+ "neutral"

form-L

A pK value (Fig.

2).

to its

It will

initial : the

c551 does not

value

of 7. It

is

not

the

for

is

when pH decreases. cytochrome

this

restored

case with

above pH % 9.7,

690 nm band disappears reappear

determined

the 690 nm band

pH in -P. aeruginosa for this cytochrome.

be given

form

t 0.1 has been

be noted-that

totally

decreasing

"alkaline"

of lz

it

transition when pH goes

-P. aeruginosa is absent

New bands c551 spectrum.

are

back

cytochrome at pH % 11 but

also

Thus,

observed

with

no pK value

can

DISCUSSION By type

comparison

cytochromes

with have

mitochondrial

a significantly

c-type lower

528

cytochrome, molecular

some weight

(Mr

bacterial 9 000)

cbut

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

9 0 O-0 c \0

Vol. 113, No. 2, 1983

29nm 0.9

(2)

lfi-

pa 9Onm - 0.12

\ 080-+To

\.

1

Q77 Fig.

to eucaryotic from

sulfate

cant

cytochrome

only

are

is

Moreover, c SS3 present pK value

it

appears

(13,14)

appears

potential

linked

to an increase

sulfur in the

1. It

worth

230 mV, though

the

that

is

similar

been isolated

have

axial

of the stability

of sulfur

the redox

heme ligands vulgaris

cyto-

to the

the Fe-S bond

atom to donate

In

pH.

It has

of the Fe-S band. related

po-

are

of increasing

can be directly in

that

and g.

to the effect

atom ; a decrase ability

noting

D. desulfuricans

resistance

a correlation

iron atom, with a stabilization potential. This correlation but it is not excluded that Recently

cytochromes

is

as a measure

that

of the methionine

of c-type

c-type

in Table

by about a strong

power

cytochromes

c5S1 the midpoint

cytochrome c5S3 and -D. desulfuri-D. vulgaris A comparison between some properties of monohemic

given

separated

chromes

lues

pH

bacteria,

c,-S~(~SD).

identical. fact,

cytochrome

two monohemic

reducing

cytochromes

been shown

1 11

cytochromes.

Up to now,

tentials

I

2 - Effect of pH on the absorbance of the 690 nm band (1) (right-hand scale)and of the 529 nm band (2) (left-hand scale) of 35 nM cytochrome c553(550) in 0.05 M sodium phosphate + 0.05 M sodium borate buffer.

in the case of Pseudomonas

c-type

, 9

donor

length

electrons

is to the

of the Fe(II1) state znd a decrease in redox seems to agree with the results given in Table I, other factors can govern the redox potential va-

cytochromes. high

c (Senn,

resolution H.,

lH NMR studies

Guerlesquin,

F., 529

on these

Bruschi,

bacterial

M. and Wtithrich,

monohemic K.,

per-

Vol. 113, No. 2, 1983

BIOCHEMICAL

TABLE I - Comparison

AND BIOPHYSICAL

between some properties

Q. desulfuricans Norway Wochrome c553(550) (6, this work) Molecular

weight

of monohemic c-type

0. vulgaris Hildenborough cytochrome c553

100

9

EA (V)/NHE at PH 7

0.04

PK

10.3 -+ 0.1

10.9 -+ 0.1

6.6

8.0

Isoelectric

point

"split a" band (reduced form)

sonal

between

"small"

bacterial

tionary

stage

tochondrial

the

oxidized

cytochromes do not show the

cytochrome

0.25

a different and the reduced

c which same type

probably

(1, 12)

12 318

+- 0.02

+ - 0.01

0.25

9.3 4.7

no

has indicated

Horse heart cytochrome c

9 000

+ - 0.01

0.02

yes

communication)

methionine

0.02

cytochromes

P. aeruginosa cytochrome cs5, (7,8, this work)

(3,4)

9 200

-+

RESEARCH COMMUNICATIONS

10.05

no

chirality form.

at the It

correspond

of methionine

no

sulfur

seems that

these

to an early

coordination

atom of evolu-

as the mi-

c.

REFERENCES 1. Dickerson, R.E. and Timkovich, R. (1975) in : The Enzymes (Boyer, P. ed.) pp. 397-457, Academic Press, New York. 2. Henderson, R.W. and Rawlinson, W.A. (1956) Biochem. J., 62, 21-29. R. and Bruschi, M. (1982) J. Electro3. Bianco, P., Haladjian, J., Pilard, anal. Chem., 136, 291-299. 4. Bertrand, P., Bruschi, M., Denis, M., Gayda, J.P. and Manta, F. (1982) Biochem. Biophys. Res. Comm., 106, 756-760. 5. Bruschi, M., Hatchikian, C.E., Golovleva, L.A. and Le Gall, J. (1977) J. Bacterial., 129, 30-38. 6. Fauque, G., Bruschi, M. and Le Gall, J. (1979) Biochem. Biophys. Res. conlm. ( 86, 1020-1029. 7. Moore, G.R., Pettigrew, G.W., Pitt, R.C. and Williams, R.J.P. (1980) Biochim. Biophys. Acta, 590, 261-271. 8. Ambler, R.P. (1963) Biochem. J., 89, 341-349. 9. Nicholson, R.S. and Shain, I. (1964) Anal. Chem., 36, 706-723. 10. Bond, A.M. (1980) Modern Polarographic methods in Analytical Chemistry, Marcel Dekker, New York. 11. Le Gall, J., Bruschi-Heriaud, M. (1968) in K. Okunuki, M.D. Kamen and J. Sekuzu, Structure and Function of cytochromes, Univ. of Tokyo Press and Univ. Park Press, p. 467. 12. Barlow, G.H. and Margoliash, E. (1966) J. Biol. Chem., 241, 1473-1477. 13. Moore, G.R. and Williams, R.J.P. (1977) FEBS Letters, 79, 229-232. 14. Cookson, D.J., Moore, G.R., Pitt, R.C., Williams, R.J.P., Campbell, I.D., Ambler, R.P., Bruschi, M. and Le Gall, J. (1978) Eur. J. Biochem.,

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