Vol. 90, No. 4, 1979 October

BIOCHEMICAL

RESEARCH COMMUNICATIONS

AND BIOPHYSICAL

Pages

29, 1979

Histidine

at the Active Lipoamide

Centre

of Chemistry

Carleton

Received

August

Heart

Dehydrogenase

D.M. Templeton Department

of Pig

1085-1090

and C.S.

Tsai

and Institute

University,

Ottawa,

of Biochemistry

KlS 586

Canada

6,1979

SUMMARY Dye sensitized photooxidation of one or more histidine residues in lipoamide dehydrogenase adversely affects enzymic activity with lipoamide as acceptor. Other enzymic activities are relatively unaffected. Evidence is presented that phosphorylation of a comnon histidine residue enhances activity with lipoamide, possibly by stabilizing an imidazolium cation. The magnetic circular dichroism spectra of reduced enzyme species display an A term attributable to charge transfer from a thiolate anion to an imidazolium cation. Upon photooxidation this term disappears. INTRODUCTION Pig heart EC 1.6.4.3)

lipoamide reduces

physiological a mixed

a number

substrate,

at a redox

(2,3)

have proposed

unidentified

Reaction

base.

structure

of which

of previously

suggested

(4),

recently

although

the base has been base of the the

charge

pig

appears

active

disulfide

proceeds

via

a topic

structures

demonstrated enzyme acceptor

unique

a mechanism

still

oxidoreductase,

and two-electron

(1).

these

this

reaction

the

half-reduced

in binding

EH2 form

charge

the thiolate

evidence which

an of the enzyme,

The most likely

EH2 form of the 5. fl

residue,

as

and

involving

thiolate-to-FAD

Here we present

The

Williams

of much discussion.

is a histidine stabilizing

amongst

for

of the (5).

substrates

of the enzyme.

involves

stabilization

heart

transfer

is

(NADH: lipoamide

of one-

lipoamide

disulfide

coworkers

the

dehydrogenase

enzyme by

that also

transfer

the

serves

catalytic as

of EH2.

MATERIALS AND METHODS Pig

heart lipoamide dehydrogenase was obtained from Sigma Chemical Co. or Boehringer-Mannheim (Diaphorase, grade I) and desalted on a P-2 column (0.9 x 15 cm) prior to use. Rose bengal (80%) was from Scientific, and methylene blue chloride (84%) was from Anachemia

(Type III) Bio-gel Fisher

0006-291X/79/201085-06$01.00/0 1085

Copyright All rights

@ 1979 by Academic Press, Inc. in any form reserved

of reproduction

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phosphoramidate was synthesized according to Chemical Co. Monopotassium Stokes (6). Photolysis was performed on 2.5 mg ml-' enzyme solutions in potassium phosphate buffer (0.010 M pH 7.0) in the presence of sensitizer at % 0.6 optical density units at the absorption maximum. Illumination was at 20 cm from a 200 watt photo-flood bulb, sample thermostated at 25.0 + O.l'C. Dissolved O2 was measured with a YSI model 57 Dissolved Oxygen Meter in an apparatus adapted from Westhead (7). Purification was on Cellex-T anion exchanger for the rose bengal experiments and by dialysis against the buffer in the methylene blue experiments. Amino acid analyses were performed on Beckman 119 BL or Durrum 0500 automatic amino acid analyzers following overnight hydrolysis Phosphorylation was --in vacua in 6N HCl at llO°C. initiated by addition of 2.5 mg of lyophilized protein to 1.00 ml of 40 mg ml-' phosphoramidate in phosphate (0.010 M) or borate (0.20 M) buffers at pH's in the range 5.7 to 9.0. Samples were withdrawn for immediate assay or filtered through the Bio-gel P-2 column. Reaction was at room temperature with continuous stirring. Spectrophotometric assays of enzyme activities were initiated by addition of 2-5 pg of enzyme to a final volume of 1.00 ml in phosphate buffer (0.010 M, pH 7.0, 25 C) containing 50 uM NADH and oxidized substrate (lipoamide, 250 PM; thio-NAD+, 125 uM; K3Fe(CN)&, 250 PM; or 2,6-dichloroindophenol, 25 PM). Magnetic circular dichroism spectra were obtained at 47 kG on an instrument built by Dr. B.R. Hollebone of this department, as previously described (8). Absorption spectra were obtained on a Cary 14 spectrometer. RESULTS AND DISCUSSION Illumination thiazine

dye,

FAD, without loss

methylene changes

dye,

destroyed,

rose

blue,

residues bengal,

accompanied (Table

dehydrogenase results

in any enzymic

of two histidine

xanthine

activity

of lipoamide

1).

by a specific In neither

in uptake

case

this

methylene

presence

of the

of 2 moles

activities.

following

replaces

in the

Amino

blue,

of O2 per mole of

acid

treatment.

analysis When the

5 histidine

decrease

in

is there

any change

cationic

reveals anionic

residues

the lipoamide-linked in the

are dehydrogenase

bound

flavin

Table 1: Modifications of enzyme activity after rose bengal sensitized photooxidation or phosphorylation. Values are expressed as percent of a control enzyme and are the average of 3 and 7 experiments respectively. Phosphorylated samples were filtered on the Bio-gel P-2 colunm at 15 min. % Control Acceptor

Substrate

Photooxidized

Activity

remaining

f S.E.

Phosphorylated

Lipoamide

37+5

280+80

thio-NAD+

68*7

1046

K3F'dCN)6

122?36

73232

2,6-dichloroindophenol

130*25

72*30

1086

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7.c-500 E J ;250

0

30

60 TIME

so

(min.)

Enzyme activity measured by rate of reduction of lipoamide after a ItIon of phosphoramidate at time = 0. 0 - 0, control (no phosphoramidate added); 0 - 0, native enzyme in 40 mg ml-'phosphoramidate in phosphate buffer (0.010 M, pH 7.0); A - A, same as 0 - 0 with addition of hydroxylamine to 1 mM at the arrow; I -m, addition of 40 mg ml-' phosphoramidate to the rose bengal photooxidized enzyme, in the phosphate buffer.

%I==

absorption

spectrum

FAD cofactor in the

to direct

necessary

substrates

are

Williams

for

less

active

the

reduction

may also

requirement

pig

heart

reaction

stabilize

a rose

a thiolate

of the enzyme with

in a transient

activity

(Figure after

retention reaction

several

1). gel

filtration

of an increased (Table

phosphorylation

1). is

Again

enhancement

is not

dependent

involvement

of a histidine

out

this

residue

of lipoamide

dehydrogenase

assay, is

is

suggested.

1087

the modified shows

specific

for

to occur

of a group

significant the

lipoamide

when the

of 5.7 to 9.0.

upon protonation

consistent

phosphoramidate

has been found

in a pH range

are

reagent

by immediate

activity.

lipoamide.

residue.

of the phosphorylgroup,

followed

functions

on partial

histidine

enhancement

The enhancement carried

substrate

formed

base

which

The above results

(4).

the phosphorylating

lability

of a catalytic

enzymes,

anion

Other

such residue(s).

disulfide

sensitive

fold

Despite

the

activity.

presence

and cr.

with

bengal

for

the

of the enzyme to the EH2 level

Reaction

enzyme,

the

difference

of one or more histidine

dehydrogenase

have suggested

of the bound

The charge

sensitization

in their

in the

such a base being

results

bengal

(2-5)

insensitivity

photodegradation.

lipoamide-linked

of both

acceptor

Such a residue

rose

demanding

site

indicating

or sensitized

and coworkers

as a proton

with

illumination,

two dyes may allow

residues

at the

after

within

Phosphohistidine

Since this

the range, displays

Vol. 90, No. 4, 1979

BIOCHEMICAL

the instability

phosphoric

group

is

with

of other

labile,

and readily

phosphorylation

phosphoramidate The active in

its

centre

role

this

factors

regard,

initially

phosphate

The visible

When native

N(1) group

%

is

a proton

thiolate

return

histidine

circular are

kinetic the

dichroism

identical

an A term appears

of (Figure

1).

and is

involved,

cation. activity

is noted.

produces

Consistent

failure

to control

as the

to N(3)

1).

stabilization If

(10)

phosphate

enzyme

imidazolium

of histidine

enzymes

(Figure

the observed

(3,4).

et al.

and magnetic

reduced,

The introduced

photooxidized

in

resultant to the

transfer

and photooxidized enzyme

of the

of Hultquist

absorption

of unmodified

the

(9).

is

by Williams

may contribute

phosphorylates

subsequent

histidine

discussed

the work

amides

RESEARCH COMMUNICATIONS

by hydroxylamine

the activity

may stabilize

Several In

displaced

base must accept

catalytic

phosphorylation

acid

of a catalytic to modify

AND BIOPHYSICAL

with

time.

Phosphoramidate product,

but

thermodynamic

product.

(MCD) spectra

in the oxidized

at 18, 180 cm-'

state.

(Figure

2(a)).

5.0 *

2.5 0

16,000

20,000

35,000

30,Ol

v(cm-11

(part a) and visible (part b) spectra of native (0 - 0) and photooxidized (0 - 0) enzymes. All spectra at 25oC in 0.010 M

MCD

%?%&a1 phosphate five-fold

buffer, excess

pH 7.0. of NADH.

obtained

anaerobically

1088

following

reduction

with

a

Vol. 90, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL

-oeo5o.

RESEARCH COMMUNICATIONS

la,000

19,000

Y (cm-') Expansion of the 17,000 EP-FP gure 2(a), obtained at higher rose bengal photooxidized enzyme. This

term

is

in reduced

absent

when free

and oxidized

MCD study

will

degenerate

excited

and thus

the A term

in a charge

to the EH2 level features

state

necessitates anion residue

is

or intensity assignment

stablized sensitive

from

spin

Formation

of the of another

by charge

transfer

to photooxidation.

ring

details

feature

system

degeneracy

for

this

A term

(2). to the

acceptor.

1089

from

electrons

anion

upon reduction

enzyme.

However, redox

The absorption

with is

from

insensitivity

state

We suggest

3(b)

a

C, symmetry,

as arising

complexation In Figure

arises

of unpaired

been interpreted

transfer

chromophore

of the

has only

of a thiolate

likely

have then charge

Full

The A term

has been considered

flavin

The MCD of the flavin

similar.

The flavin

arise

of the EH2 species

of the energy

is

elsewhere.

complex.

thiolate-to-oxidized

reduced.

enzyme

(11).

must

transfer

FAD is

native

be published

to 19,000 cm-I region of the MC0 spectra gain. (a) reduced native enzyme, (b) reduced

of the

that

the

a catalytic shown

flavin thiolate histidine

the expanded

Vol. 90, No. 4, 1979

A term

region

enzyme

(Fig.

a shoulder enzyme

BIOCHEMICAL

when the photooxidized 3(a))

no A term

in the

this

visible

shoulder

is

the flavin

after

spectra

to be intact

enzyme

results

(Figure

loss

In conclusion, active

site

of this

residue

heart

and phosphorylation The residue via

charge

appears

transfer

2(b)

loss

band.

shows

It

thiolate

lipoamide selectively

to be involved

complexation

in

loss

show

of the charge

differences

in the reduced

has been

identified

Sensitized dehydrogenase increases

this

stablization

in the reduced

spectra

in the MCD

dehydrogenase.

residue

these

histidine.

residue

decreases

In the native

Differences

stabilizing

histidine

of

has been attributed

Therefore

structural

to native

the absence enzyme.

However

of histidine.

lipoamide

In contrast

photooxidized

photooxidation.

of the

of the

also

of the

additional

selectively

reduced.

Figure

transfer

a catalytic

of pig

is

to the MCD A term.

from

2) indicate

accompanying

seen.

corresponds charge

feature

enzyme

spectrum

to a thiolate-to-flavin

transfer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at the

photooxidation activity, activity.

of a thiolate

residue

enzyme.

ACKNOWLEDGEMENTS The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada. Several amino acid analyses were kindly supplied by Dr. M. Yaguchi of the National Research Council of This communication is taken in part from the Ph.D. thesis Canada. submitted by DMT to the Faculty of Graduate Studies and Research, Carleton University. REFERENCES

1. 2.

Massey, V. (1963). The Enzymes, vol. VII, P.D. Boyer, Ed., pp. 275-306, Academic Press, New York. Williams, C.H., Jr. (1976). The Enzymes, vol. XIII, pt. c, P.D. Boyer, Ed., pp. 90-174, Academic Press, New York.

Jr, (1976), J. Biol. Chem. 3, 3956-3964. (1976). J. Biol. Chem. 251, 3553-3557. C.H. Jr. (1979). J. Biol. Chem, 254, 852-862.

3. 4.

Matthews, R.G., and Williams, C.H. Thorpe, C., and Williams, C.H., Jr.

5.

Wilkinson,

6.

Stokes,

7.

Westhead,

E.W.

8.

C,H.,

9.

Langford, in press. Schneider,

10.

Hultquist,

D.E.,

11.

Holmquist,

B.,

K.D.

and Williams,

H. (1893) Am. Chem. J. 15, (1964)

198-214. 4, 2139-2144.

Biochemistry

Hollebone,

B.R.

F. (1978) Angew. Moyer, and Vallee,

and Vandernoot,

Chemie.,

R.W., B.L.

Int.

and Boyer, (1978).

1090

T.

Ed. II, P.D.

(1979).

Adv.

583-592.

(1966). Biochemistry

Methods

Chem. Ser.,

Enzymol.

49(G),

5,

322-331.

149-179.