Vol. 169, No. 3, 1990 June 29, 1990

Im

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 1235-1241

SPlEmA OF FERRIC f-zmcmmg* FRoHmsTBbMsoF ILYWSOXIDMS AT IN TEnPERATlJREANDTHErR TlMPERATURE

A-DA= Tetsuhiko

Yoshimuraal, Hidekazu

aThe

Shinnichiro Suzukib, Takamitsu Iwasakic, and Sohsuke Shidarad

DHWiDEHCE

Kohzumab,

Environmental Science Institute of Hyogo Prefecture, Yukihira-cho, Suma-ku, Kobe 654, Japan

bInstitute of Chemistry, Osaka University, cNagoya

University

dDepartment and Sciences, Received

AND BlOPHrSlCAL

College Toyonaka,

of General Osaka 560,

Education, Japan

of Foreign Studies, Nisshin-cho, Aichi 470-01, Japan

Aichi-gun,

of Environmental Science, Faculty of Integrated Hiroshima University, Naka-ku, Hiroshima 730,

May 21,

Arts Japan

1990

Sumusry: The EPR spectra at low temperature (6 K) and their temperature dependence (lo-93 K) for five ferric cytochromes c' isolated from chemoheterotrophic bacteria, , Achromobacter xylosoxidans NCIB 11015 (formerly Alcaligenes sp. NCIB 11015), GIFU 543, GIFU 1048, GIFU 1051, and GIFU 1764 are reported. The EPR spectral results indicate that the ground state of the heme iron(II1) of cytochromes c' from these chemoheterotrophic bacteria can appear to be in an admixed spin state which consists of predominant S = 512 with a slight S = 312 character. The EPR spectra were compared with those for ferric cytochromes r' from photosynthetic bacteria and the other ferric hemoproteins. 01990

Academic

Press,

Inc.

Cytochromes chemosynthetic

c'

of about

however,

they

of other

-c-type

admixture

Most

daltons.

d.

ground

cytochromes

from

belong

to c-type

and magnetic magnetic

which

consists

(S = 3/2) by X-ray

cytochromes; unlike

properties

those

of ferric

pH have been electronic

of quantum

and a high-spin

crystallographic

bacterium

and

identical

properties

heme-iron

state,

should

with

as due to the

a photosynthetic

1 To whom correspondence

dimers

at neutral

has been established c'

of photosynthetic

bacteria

of an intermediate-spin It

proteins

The unusual

&

a variety

of them are

These

photosynthetic

at the

from

spectroscopic

cytochromes.

by Maltempo

configuration

(4,5).

14000

from

isolated

(l-3).

have unusual

c'

interpreted

been

bacteria

subunits

cytochrome

have

Rhodospirillum

mechanical (S = 5/Z)

studies

state

of the

molischianum

be addressed.

1235

CXMfi-291X/90 $1.50 Copyright 8 1990 by Acad2mic Press. Inc. All rights of reproduction in any form reserved.

Vol.

169,

that

No.

3, 1990

BIOCHEMICAL

the heme fifth

ligand

is a histidine

coordination

position

surrounded

vacant

Although

X-ray

(6).

chemoheterotrophic environments those acid

bacteria

c'

sequence of these We have recently

by hydrophobic

has so far c'

cytochromes

properties

RESEARCH

and that amino acid

analysis

is 2' from

the heme considered

from the comparison

to resemble of the amino

(7).

demonstrated that there are the differences and chemoheterotrophic

and ligand binding characters

differ

between

bacteria

(2,3,8-14).

suggest that the heme environments also delicately -C’.

residues

for cytochromes

have been reasonably

c'

COMMUNICATIONS

the sixth

not been performed,

from -R. molischianum,

cytochromes 2' from photosynthetic spectral

BIOPHYSICAL

residue

crystallographic

in the cytochromes

in cytochrome

AND

in the

These findings

for each cytochrome

In the present communication, we report the EPR spectra at low

temperature and their isolated

temperature dependence for five

from chemoheterotrophic

bacteria,

such as Achromobacter xylosoxidans Ach. xylosoxidans

11015)2(2),

Ach. xylosoxidans xylosoxidans

denitrifying

NCIB 11015 (formerly

GIFU 543 (3), &

cytochromes 2'

Alcaligenes

xylosoxidans

GIFU 1051 (8) and non-denitrifying

bacteria sp. NCIB

GIFU 1048, and

bacterium, &

GIFU 17643 and compare them with the spectra for ferric

cytochromes c'

Materials

including

ferric

from photosynthetic

bacteria

and the other ferric

hemoproteins.

and Methods

Cytochromes 5' from Achromobacter xylosoxidans NCIB 11015, GIFU 543, GIFU 1048, GIFU 1051, and GIFU 1764 were isolated and purified in a manner similar to the methods described previously (2,3,8). Cytbchrome c' solution measured was adjusted to pH 7.2 (50 mMKH2P04/Na2HP04). EPR measurementswere carried out on a JEOL RE-2X (X-band) spectrometer with loo-KHz field modulation; microwave frequencies and magnetic fields were determined with an Advantest frequency counter Model TR-5211A and an Echo Electronics NMRfield meter EMF2000A, respectively. The temperature was controlled by an Air Product LTR5500 Helitran transfer system. Other EPR and experimental conditions are described in the figure legends.

2 Alcaligenes sp. NCIB 11015 was recently Achromobacter xvlosoxidans (15,16).

identified

as a normal memberof

3 This bacterium produces no gaseous products from nitrate and nitrite, but is able to grow in the presence of nitrous oxide as an electron acceptor (9). 1236

Vol.

169, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Results and Discussion EF%spectra of Ferric of ferric ferric

cytochrome cytochromes

splitting

c'

around

parameters

together

1, the

values

side

and the

zero

g3 was also

corresponding quantum

with

those

crossing

the

to a quantum

out

percentage

that

state

The EPR spectrum characteristics

trough

of the

1

a heme-iron

ferric

I 2aoo

spin

cytochrome I)

1

field

with

g values

Gupta

of

The of 4