Properties of the covalently bound flavin of chromatium cytochrome c-552 and its conversion to 8-carboxy-riboflavin

Vol. 46, No. 3, 1972

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PROPERTIES OF THE COVALENTLY BOUND FLAVIN OF CHROMATIUM CYTOCHROME c-552 AND ITS CONVERSION TO 8-CARBOXY-RIBOFLAVIN Robert Hendriks and John R. Cronin Department of Chemistry, Arizona State University Tempe, Arizona 85281 Wolfram H. Walker and Thomas P. Singer Molecular Biology Division, Veterans Administration Hospital, San Francisco, California 94121 and Department of Biochemistry and Biophysics, University of California Medical Center, San Francisco, California 94122

Received December 27, 1971 SUMMARY. Previous work has shown that cytochrome c-552 from Chromatium contains a covalently bound flavin, which is not released by denaturation but is liberated by proteolysis or various chemical treatments and that the protein is probably attached at the 8~ position of the FAD. In the present study unambiguous evidence was obtained for 8~ substitution from (a) ESR hyperfine spectra of the flavin cation radical and (b) from identification of the flavin released by performic acid oxidation from cytochrome c--552 as riboflavin-8~-carboxylic acid. Various lines of evidence suggest that in the native enzyme a reduced sulfur may be linked to the 8~ group of the flavin, similarly to monoamine oxidase, which contains cysteinyl 8~-FAD, although the conditions required for cleavage of the flavin from the two enzymes appear to be quite different. INTRODUCTION Cytochrome c-552 obtained from Chromatiam has been shown to contain a firmly bound flavin prosthetic group (i). Flavin is not released from the cytochrome by precipitation with either trichloroacetic acid (TCA) or acid ammonium sulfate but is

released slowly in the presence of saturated urea,

or by proteolysis, exposure to alkaline pH, or treatment with p-chloromercuribenzoate (2). The flavin released by prolonged digestion with urea appears to be 8~-substituted FAD, judging from the hypsochromic shift of its second absorption band (3) but is different from the histidyl-FAD present in succinate dehydrogenase (4, 5) because the Chromatium flavin shows no pH dependence of its fluorescence between pH 3 and 7 (3). The present paper provides conclusive evidence for the 8a-methylene of FAD as the site of attachment of the protein in the cytochrome, presents a quantitative study of various methods of

1262 Copyright ©1972, by AcademicPress, Inc.

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releasing the Chromatium flavin which distinguish it from the covalently bound flavin of both Succinate dehydregenase (SD) and monoamine oxidase (MAO) (6, 7), and describe studies suggesting the presence of a reduced sulfur moiety at the

8a site (Fig. i). R

.N. N / 0 "-1~8 "y ,o "['-" 1 ~Y

X-H2C..

~

0

_

I

X =

N.--~ N I

NHRI I

I CH2C N H R I

2 Fig. i.

=-S-CH2CH

H

COR2

SD-FLAVIN

I

C0R 2

MAO- FLAVIN

Structures of covalently bound flavins.

R = rest of FAD.

MATERIALS AND METHODS Chromatium strain D cells were grown and harvested, cytochrome c-552 isolated and purified, and flavin obtained by urea treatment of the flavocytochrome as previously described (3).

Proteolytic digestion was performed under

N 2 with 0.i mg each of crystallized trypsin and chymotrypsin per mg of cytochrome c-552 either at pH 7 or at pH 7.9 in 0.i M Tris buffer for 2 to 3 hrs at 38 ° , with or without 2 mM dithiothreitol (DTT) present, as indicated in the text.

The yield of flavin liberated was based on analysis of the supernatant

obtained on precipitation with 5% (w/v) trichloroacetic acid at 0 °.

Purifica-

tion of the flavin from proteolytic digests involved chromatography on Florisil, elution with 5% (v/v) pyridine, and chromatography on phesphocellulose col1~mns (pyridinium cycle) with 20 mM pyridine acetate, pH 4.0, followed by high voltage electrophoresis in 8% (v/v) formic acid (pH 1.6). Flavin release by performic acid oxidation was achieved by treating an 88% formic acid solution of the cytochrome according to the procedure of Kimmel et al. (8).

Electrophoresis at pH 1.6 (8% formic acid) and pH 3.5 (10%

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acetic acid-l% pyridine) was run at 50 V/cm. corded with a Hitachi-Perkin

Elmer Model MPF-3

with a spectral correction accessory.

Fluorescence

spectra were re-

spectrofluorometer

8-Carboxy-riboflavin

riboflavin were synthesized according to Kenney and Walker

equipped

and 8-a-sulfonyl(9).

RESULTS AND DISCUSSION Fig. 2 shows the ESR hyperfine spectrum of the radical cation of the flavin obtained from cytochrome !-552 by prolonged exposure to a saturated urea solution at 4oc.

Prior to obtaining the spectrum,

jected to mild acid hydrolysis

the flavin was sub-

and acid phosphatase digestion to cleave the

I

lOG

Fig. 2. ESR spectrum of the cation radical of Chromatium was released by urea treatment, purified, and degraded to . . . . wiL~, ~u ~.+3 we re reco r ded (3); 20 nmoles in 6 N HCI reducea ~l a Varian E-9 spectrometer at 9.1 GHz resonance frequency, modulation amplitude, i00 KHz modulation frequency, i sec 8 min scanning time.

flavin. The flavin the riboflavin level at room temp. with 40 mW power, 0.25 G time constant, and

pyrophosphate linkage and remove the 5'-phosphate residue

(3).

The ESR spec-

trum is essentially identical both in signal width (37 G) and hyperfine structure (17 lines, 2.3 G spacing) with that of cysteinyl-8a-riboflavin, vin component of MAO (6) and differs characteristically of both normal flavins and of histidyl-8~-riboflavin

the fla-

from the ESR spectra

(i0).

For reasons detailed in previous paper s (i0, ii) this type of characteristic alteration of the ESR spectrum of riboflavin constitutes that the substitution is indeed in the 8a position.

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strong evidence

The virtual coincidence

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This further establishes

that the flavin is indeed substituted in the 8~ posi-

tion in the cytochrome. As in the case of the MAO flavin peptide chymotrypsin digestion shows a hypsochromic

(6), the product of trypsin-

shift of the second absorption

band from 372 nm to about 365 nm and a strongly quenched fluorescence

and on

oxidation with performic acid the second absorption band shifts to 352 nm, with a simultaneous

7 to 8-fold increase in fluorescence

This shift in the maximum and the enhancement dicate an electron-donating possibility

(Fig. 3 and Table II).

of fluorescence

on oxidation in-

substituent at 8e in the Chromatium flavin.

that, as in MAO, sulfur is the substituent

The

is not established with

certainty as yet, although the results in Figs. 2 and 3 are compatible with this and, further, when the flavin released by proteolytic digestion in the presence of DTT was purified, tained after electrophoresis and chloroplatinic

as given in Methods,

the homogeneous

product ob-

gave positive tests for reduced S in the I2-azide

acid tests.

I

;

I

80

I

I

448

352

I

[ 365

~j 6 0

4.0

20 f

300

f

I

4-00 WAVELENGTH

I

I

500 (rim)

Fig. 3. Fluroescence excitation spectra of Chromatium flavin. The product obtained from proteolytic digestion in ~esence of DTT without hydrolysis is shown in curve A and after performic acid oxidation in curve B but using 10% as much flavin as in A.

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In searching

for clues to the nature of the substituent at 8~ and the

linkage involved the yields of flavin liberated under well-defined have been compared

(Table I).

conditions

The data in this table are based on fluores-

cence yields, which are, of course, influenced both by the state of oxidation of the compound released and by the presence or absence of the dinucleotide. In order to correct for these factors, nucleotide,

in treatments expected to yield a di-

fluorescence was examined before and after acid hydrolysis,

and

to correct for the oxidation state of the flavin, fluorescence was measured before and after oxidation with performic acid.

(The flavin released by di-

rect performic acid oxidation of the eytochrome,

as well as authentic 8-car-

boxy-riboflavin,

yield 80% of the fluorescence

riboflavin or FMN, when concentrations nm (Tables I and II).

of an equivalent amount of

are normalized from absorbance at 450

In case of treatments which failed to release signifi-

cant amounts of flavin in trichloroacetic

acid soluble form, the residue was

subsequently treated with performic acid to show that the flavin was still bond to the denatured protein. The data in Table I and the results of other studies indicate that the flavin is linked to the protein in an acid- and heat-stable but alkali-labile linkage, which is not cleaved by reducing agents but is labile to strong oxidizing agents as well as mercurials

(2).

In addition,

zyme results in the release of 8-carboxy-riboflavin.

direct oxfdation of enOn the basis of the in-

dications given of the presence of reduced S at the 8~ position, possibilities

have been considered:

8e-thioflavin

(disulfide),

the following

a cysteine -SH may be joined to (a) an

(b) to an 8~-OH group (thioether),

(c) to an 8-for-

myl group (thiohemiacetal),

or (d) an 8-COOH group (thioester).

Possibilities

(a) and (d) are improbable,

since neither reducing agents nor NH2OH liberate

the flavin (Table I); (b) appears somewhat unlikely in view of the ease of liberation of the flavin by alkali and performic acid but has not been ruled out, while (c) still remains a possibility.

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with the ESR spectrum of cysteinyl-8~-riboflavin may further suggest that the Chromatium flavin liberated by urea also contains a sulfur substituent at the 8e-CH2, although it should be noted that certain other 8-substituted flavins not containing sulfur (8-carboxy-riboflavin, 8~-O-tyrosyl-riboflavin) yield identical ESR spectra (i0). On treatment of the cytochrome preparation with performic acid the flavin is released in excellent yield (Table I).

This behavior distinguishes

TABLE I Release of Flavin from Chromatium Cytochrome by Various Treatments

Treatment

Fluorescence yield in deproteinized supernatant a) %

Performic acid

80

0.i M KOH, 60 ° , 20 min

34

Same after performic acid oxidation

34

4 M NH20H , pH 6.1, 60 ° , 20 min

0

2 mM DTT, pH 7, 37 ° , 8 hr

0

4 mM dithionite, pH 6.9, 38 ° , 2 hr

0

Trypsin-chymotrypsin, N2, 2 mM DTT (cf. METHODS) b)

7

Same after hydrolysis in N HCI

13

Same after performic acid oxidation

81

a)This value is a function both of the amount of flavin released and of the degree of fluorescence quenching in the sample. The flavin yield cannot be directly determined from absorbance, except after purification, as in the last sample, because of the presence of interfering materials. Proteolytic digestion (last sample) appears to release all the flavin, as judged by absorbance and the purified flavin gives 80% of the fluorescence of riboflavin after performic acid oxidation. Fluorescence/0.8 may be used to calculate flavin yield in all samples in which the flavin is in the form of 8-carboxy-riboflavin. Excitation at 450 nm, emission at 525 nm. b)When DTT is omitted during digestion, the same results were obtained after performic acid, but before oxidation the fluorescence yield was slightly higher than given in the Table, probably because of partial oxidation during proteolysis.

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the Chromatium flavin from the covalently bound flavin of MAO, which is oxidized to the sulfone without being released from the peptide under these conditions (6).

The Chromatium flavin thus liberated, after dephosphorylation,

has been identified as 8-carboxy-riboflavin on the basis of its fluorescence excitation spectrum, fluorescence yield relative to riboflavin, pK a value, electrophoretic mobility, and migration in TLC (Table II).

8~-Sulfonyl-ribo-

flavin, a possibie oxidation product of an 8e-S-substituted flavin, is readily distinguishable from the product obtained by performic acid oxidation (9).

TABLE II Comparison of Chromatium Flavin Released by Performic Acid with 8-Carboxy-riboflavin

Result Criterion Chromatium flavin a)

8-Carboxy-riboflavin

Fluorescence excitation spectrum, Ima x at pH 6.0 at pH 2.0

448, 368 nm 448, 352 nm

448, 367 nm 448, 352 nm

81

84

Fluorescence yield, % of riboflavin

ESR spectrum b)

Electrophoretic

17 lines, 2.3 G spacing

17 lines, 2.3 G spacing

mobility c)

at

pH 1.6 pH 3.4

+0.2

+0.2

+i.i

+I.i

2.5

2.5

0.42

0.42

PKad) value in TLC e)

a) After dephosphorylation, d~ b) In 6 N HCI. c) The migration of FMN re 1 ative to rib o flavin is taken as + 1.0. "Determined, from the pH-dependence of the position of e2 second fluorescence excitation band. On silicic acid in N-butanol: acetic acld: H20 (4:2:2, v/v).

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ACKNOWLEDGMENTS This research was supported by grants from National Institutes of Health (AM-12908 and HE 10027) and the American Cancer Society (BC 46A). We wish to thank Dr. D. Lenniart of Varian Associates for help with the ESR experiment and Mr. R. Seng for skilled assistance. REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9. i0.

ii.

R . G . Bartsch, Fed. Proc., 20, 43 (1961). R.G. Bartsch, T. E. Meyer and A. B. Robinson, in "Structure and Function of Cytochromes," K. Okunuki, M. D. Kamen, and I. Sekuzu, eds., University Park Press, Baltimore, Md., 1968, p. 443. R. Hendriks and J. R. Cronin, Biochem. Biophys. Res. Communs., 44, 313 (1971). W . H . Walker and T. P. Singer, J. Biol. Chem., 245, 4224 (1970). W . H . Walker, T. P. Singer, S. Ghisla, and P. Hemmerich, Eur. J. Biochem., inpress. W . H . Walker, E. B. Kearney, R. Seng, and T. P. Singer, BiOchem. Biophys. Res. Communs., 44, 287 (1971). W . H . Walker, E. B. Kearney, R. Seng, and T. P. Singer, Eur. J. Biochem., in press. J . R . Kimmel, G. K. Kato, A. C. M. Paiva, and E. L. Smith, J. Biol. Chem., 237, 2525 (1962). W . C . Kenney and W. H. Walker, FEBS Letters, in press. T . P . Singer, J. Salach, W. H. Walker, M. Gutman, P. Hemmerich, and A. Ehrenberg, in "Flavins and Flavoproteins," H. Kamin, ed., University Park Press, Baltimore, 1971, p. 607. P. Hemmerich, A. Ehrenberg, W. H. Walker, L. E. G. Eriksson, J. Salach, P. Bader, and T. P. Singer, FEBS Letters, 3, 37 (1969).

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