Assignment of exchangeable proximal histidine resonances in high-spin ferric hemoproteins: Substrate binding in horseradish peroxidase

Vol. 90, No. 1, 1979 September

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12, 1979

Pages 36-41

ASSIGNMENT OF EXCHANGEABLE PROXIMAL HISTIDINE RESONANCES IN HIGH-SPIN

FERRIC HEMOPROTEINS:

SUBSTRATE

BINDING IN HORSERADISH PEROXIDASE* Gerd N. La Mar and Jeffrey Department

Received

July

S. de Ropp

of Chemistry, University of California Davis, California 95616

25,1979 SUMMARY

Single-proton, exchangeable resonances have been detected in the high spin ferric hemoproteins, met-aquo myoglobin and horseradish peroxidase, which can be assigned to the proximal histidyl imidazole by virtue of their very large hyperfine shifts. While this proton is relatively labile in myoglobin, it is exchangeable in HRP only at extreme pH values, indicating a buried heme pocket. The insensitivity of the imidazole peak of HRP to substrate binding argues against direct interaction of imidazole and substrate. Proton of detailed large

nmr spectroscopy structural

hyperfine

any appended sixth

ligand

(1,Z).

based

proximal culty

lating

compound

work

studies

unambiguous models

(7).

*Supported by grants UCD NMR Facility.

from

endogenous

histidine

However,

until

the need

importance center.

the National

for

several

Copyright AN rights

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

36

and

probes based

heme peak

assign-

(4-6),

axial

ligand

resonances,

because

recently, assignments

(i.e.

diffiof any

of such resonances

interactions

The iron-histidine of Health,

of the

the absence

bond

in moduis

HL-16087,

Abbreviations used: DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate; HRP, horseradish peroxidase; metMb, met-myoglobin; IPA, indole 0006-291X/79/170036-06$01.00/0

group

or an exogenous

assignment

of ligand-iron

Institute

due to the

as structural

reported

primarily

and,

a wealth

the heme prosthetic

recently

have been

on the

iron

primarily

and probable

although

assignment

of the

site

proximal

have been rare

by the proposed

the reactivity

(3),

relying

in making

for

resolution

deuteration

imidazole),

underscored

active

has provided

use of heme resonances

ready

histidyl

characterized is

The dominant

on selective

Structural

on their

can be observed

to their

on model

ments

which

hemoproteins

such as the ubiquitous

can be attributed largely

information

shifts ligand

of paramagnetic

propionic

central and the

acid

Vol. 90, No. 1, 1979

to the model

for

gen bonding

BIOCHEMICAL

cooperativity

between

in hemoglobins

the proximal

bony1

has recently

(9) -

In the case of horseradish

has been

At this

to act

is

sufficiently

strongly

ments

shift

(2,3)

have been

myoglobins

ferric

models

reported

in hydrogen

deoxy

with

spin

deuterium

myoglobins

imidazole (10).

resonances

only

this

in

proton

to exhibit

in 2H20.

is both

a large

Successful

and hemoglobins

car-

in hemoproteins

histidyl

imidazole

iron

backbone

to substrates

N,H because

to the unpaired

exchangeable

mechanism

bonding

of these

to the pyrrolic

for

control

of hydro-

(7)

hyperassign-

and met-cyan0

(11).

To date, spin

and is

and a peptide

the proximal

assignment

coupled

RESEARCH COMMUNICATIONS

and the variability

imidazole

as a general

as a donor

restricted

(8),

peroxidase,

unambiguous

time,

hemoproteins

fine

histidyl

been proposed

proposed

AND BIOPHYSICAL

however,

relatively

hemoproteins

and the very

little

nmr work

due to a combination

substantial

linewidths

has been reported

of the observed

absence even

for

on high-

of any suitable heme methyls

(6, 12). In view

of the

catalases

(13)

governing

their

investigating

of this

and oxygenases unique their

successful

observation

horseradish

peroxidase

zole

importance

(14),

reactions,

and the

state

possible

such assignments

structure-function

role would

relationships.

of exchangeable which

oxidation/spin

can only

resonances arise

from

in heme peroxidases, of the histidine

be highly We report

in met-aquo the

proximal

useful here

myoglobin histidyl

in in

on the and imida-

NIH. METHODS

Sperm whale myoglobin (Type II) and horseradish peroxidase, HHP, (Type VI, RZ = 3.1-3.2) were purchased from Sigma as salt-free, lyophilized powders. The nmr spectra were found insensitive to any further purification (15). Indole propionic acid, IPA, was obtained from Sigma and used without further purification. Solutions were prepared in either 2H20 or 90% H,f/lO% 2H20 (2-3 mM) 0.2 M in NaCl. The pH was adjusted using 0.2 M NaOzH and HCl. pH readings (Beckman 3550 pH meter, Ingold micro-combination electrode) are uncorrected for isotope effect in 'H,O. 360 MHz nmr spectra were recorded on a Nicolet NT-360, utilizing lOK-100K transients with 8K points over 80 KHz bandwidth. The 90" pulse (8.7 us> was centered on the solvent resonance which was suppressed by a saturating pre-pulse. Apodization introduced a negligible 20 Hz line broadening. Chemical shifts are given in ppm, referenced to 2,2-dimethyl-2-silapentane-5-sulfonate. DSS.

37

BIOCHEMICAL

Vol. 90, No. 1, 1979

100

80

60

Chemical

Fig.

Proton

1:

AND BIOPHYSICAL

NMR Spectra

Shift

40

, in

ppm

::,

of Sperm Whale Met-aquo

Low-field portion of the 360 MHz proton in A. 99.9% 'H20; B. 90% H,O, 10% 'H,O. assigned by deuterium labeling (6).

RESEARCH COMMUNICATIONS

20

w

Myoglobin

nmr traces of 2mM protein, pH 6.2, 35°C The peaks common to A and B have been

RESULTS The proton myoglobin between

nmr spectra

in 'H,O the

of the

and H,O at 35°C are

two traces

is

100 ppm in H,O which

is

The low-field

proton

the

except

for

much improved

2.

that before

returned

in C is also readjusting

clearly

The effect

methyl

peaks

exchangeable

dictating

of IPA binding 2, which

in the

in a manner

peak

at 96 ppm is

the

The only proton

difference

resonance

20-50

same as reported

ppm region

in C of Figure

shown)

if

the

C differs that

on the

it

similar

at

is

an exchangeable

to that

38

first

reported

While

(16)

detection

of

to

identical raised

to to 11.4

the peak at 96 ppm proton.

of HRP at 35'C

in A.

earlier

lowered

A trace

pH is

illus-

H,O and *H20.

was first

from A in that

to that

unaffected.

and the

neutral

2.

sample

nmr spectrum

can be compared

shift

are

at 96 ppm in both

shown

Trace

1.

single

the pH of the protein

(not

to 7.0.

has disappeared,

in D of Figure

after

to 7.0 is

obtained

of a broad

The spectra

intensity

as in A run

in Figure

met-aquo

of HRP in *H,O and H,O at 35'C are

resolution

a new peak of single-proton

of sperm whale

the *Hz0 solution.

nmr spectra

in A and B of Figure

3.1 and then

in

region

compared

appearance

missing

trated

The same trace

downfield

is

illustrated

the four

previously

heme

(16),

the

Vol. 90, No. 1, 1979

BIOCHEMICAL

Chemical Fig.

2:

Proton

NMR Spectra

of

AND BIOPHYSICAL

Shift

,

in

Horseradish

ppm

RESEARCH COMMUNICATIONS

v*

Peroxidase

Low-field portion of the 360 MHz proton nmr traces of the resting state of HRP, pH 7.0, 35"C, in A. 90% H20, 10% 'H,O; C. same as in A except 99.9% 'H,O; B. after first lowering the pH to 3.1; D. in 99.9% H,O after adding a 15-fold excess of indole propionic acid. The dotted lines indicate the shift change induced by IPA binding. DISCUSSION

The appearance *H,O solution

of metMbH,O

strongly

coupled

8~ spin

delocalization

systems mitted

(3),

been

it

to the

the proximal

to the

histidyl

proton

(Figure ferric

highly

proximal

the

single

which is

characterized

influencing

of the

iron-histidine

but

not

dominates improbable

imidazole which

1) dictates ion

histidine

could

peak at 100 ppm in H,O but not

the that

peptide N,H. shed bond.

that belonging

contact the NH.

iron

spin

Hence this models

that

an exchangeable

to the heme.

on any unique

The fact

39

is

shifts

No suitable 1 ight

it

in

in high density

role

Due to the spin

ferric

can be trans-

peak must for

proton

arise

metMbH,O

from

have

of the protein

in

the peak at 100 ppm is ab-

Vol. 90, No. 1, 1979

sent

immediately

BIOCHEMICAL

after

access

of water

site.

NMR evidence

sented

recently

dissolving

protein

to the heme cavity for

in the

considerable

latter

where

solvent

that

2.

for

iron

that

isotope is

resonances

ress

laboratory.

Previous

studies

Although both

in HRP.

has been pre-

at neutral

this

are

carried

similar

state

(3.1

with

'Hz0

its

peak

assignment

evidence

however,

more buried

in H,O,

of this

for

of HRP (17).

'H,O,

values

out

shift

supports

Hz0

pH, as shown

peak exchanges

additional

in

in both

the pH to extreme

to

to the the pres-

The stability

is

and less

of ligation

of the

heme in both

on the

is known and esr

is

stabilized

imidazole

of the traces

B or C in Figure

reveals

while

of the

has been

indicative solvent

of a acces-

(20)

binding

that

is

analysis iron

interpreted

their

stereoto the

argue also

against

found

await

Such a study

is

paramagnetic in terms

of a hydrogen-bond heteroatom.

of the HRP-IPA

by

of the hyperfine

in HRP must

(10)

substrate

does influence

40

evidence

HRP-induced

by formation

that

to be coordinated

proteins.

N,H and the 2 with

suggest

structure

stereospecific

where

substrate

also

A more detailed

degree

ture

histidyl

water

nmr (19)

(21).

substrate

binding

two proteins

A five-coordinated

of a proton-donor the

is

weeks

1 strongly

exchange

in these

tion

that,

active

observed

The very

considerable

(18),

to the

ment of all

the proximal

or exposed

peak

pH changes

and provides

Raman spectroscopy relevant

these

in the resting

N,H shift

water

in our

ready

in myoglobin.

in met-myoglobin

resonance

the

chains

occurs,

in Figure

must be similar.

a coordinated

shifts

towards

in HRP which

The similar chemistry

with

of heme side

changing

unaffected.

histidine

NIH bond

than

If

imidazole,

ence of a proximal

upon

2).

metMbH20

N,H of a coordinated

sible

loose

several

denaturation

at 96 ppm remains

heme pocket

a relatively

single-proton

However,

(C in Figure

observed

of this

consistent

motion

even after

reversible

and disappears the peak

is

(6).

in A and B of Figure or 11.4),

in2H20

and/or

In the case of HRP, a broad, 2H,0,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

assignin prog-

relaxaof a strucbetween Comparison

complex, given in D,

the heme resonances,

the

Vol. 90, No. 1, 1979

hyperfine plete

shift

of the

insensitivity

not

only

the

substrate

BIOCHEMICAL

argues

exchangeable

of the proximal strongly binding

A more detailed on extensive

isotope

be presented

elsewhere.

against

labeling

RESEARCH COMMUNICATIONS

N,H peak

at 96 ppm is unchanged.

histidyl

imidazole

such hydrogen

is more likely description

AND BIOPHYSICAL

bonding

on the distal

of the binding of heme resonances

N,H peak

side

(lo),

but

com-

to IPA binding suggests

that

of the heme.

of the substrates sensitive

This

to HRP based

to IPA-bind

ing

wi 11

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Morrow, J. S. and Gurd, F.R.N (1975) CRC Crit. Rev. Biochem. 2, 121-189. La Mar, G. N. (1979) Magnetic Resonances in Biology, Shulman, R. G., ed., Academic Press, N.Y., in press. La Mar, G. N. and Walker-Jensen, F. A. (1979) The Porphyrins, Dolphin, D., ed, Academic Press, N-Y., 61-157. Mayer, A., Ogawa, S., Shulman, R. G., Yamane, T., Cavaleiro, J.A.S., Rocha Gonsalves, A.M.d'A., Kenner, G. W., and Smith, K. M., (1974) .I. Mol. Biol. 86, 749-756. La Mar, G. N., Budd, D. L., Viscio, D. B., Smith, K. M., and Langry, K. C., (1978) Proc. Nat. Acad. Sci. U.S. 2, 5755-5759. La Mar, G. N., Budd, D. L., Smith, K. M. and Langry, K. C., (1979), J. Am. Chem. Sot. in press. La Mar, G. N., Budd, D. L., and Goff, H., (1977). Biochem. Biophys. Res comm. 77, 104-110. Perutz, M. F., (1976) Brit. Med. Bull. 32, 195-208. Valentine, J. S., Sheridan, R. P., Allen, L. C., and Kahn, P. C., (1979 Proc. Nat. Acad. Sci. U.S. 76, 1009-1013. Schejter, A., Lanir, A., and Epstein, N., (1976) Arch. Biochem. Biophys 174, 36-44. Sheard, B., Yamane, T., and Schulman, R. G., (1970) J. Mol. Biol 53, 35-48. Morishima, I., Ogawa, S., Inubushi, T., and Iizuka, T., (1978) Adv. Biophys. 11, 217-245. Saunders, B. C., (1973) Inorganic Biochemistry, Eichhorn, G. I., ed, Elsevier, Amsterdam, 988-1021. Gunsalus, I. C., Meeks, J. R. Lipscomb, J. D., Debrunner, P., and Mtinck, E. (1974), Molecular Mechanisms of Oxygen Activation, Hayaishi, O., ed, Academic Press N-Y.. 561-613. Shannon, L. M., Kay, E.; and Lew, J. Y., (1966). J. Biol. Chem. 241, 2166-2172. Morishima, I. and Ogawa, S., (1979) J. Biol. Chem. 254, 2814-2820. Dunford, H. B. (1979) Adv. Inorg. Chem., in press. Takano, T., (1977) J. Mol. Biol. 110, 537-568. Lanir, A. and Schejter, A. (1975), Biochem. Biophys. Res. Comm. 62, 199-203. Vuk-Pavlovic, S. and Siderer, Y., (1977),Biochem. Biophys. Res. Comm. 2, 885-889. Spiro, T. G., Strong, J. D. and Stein, P. (1979), J. Am. Chem. Sot. I&, 2648-2655.

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