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

Vol. 46, No. 3, 1972 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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-chloromercuri-

benzoate (2). The flavin released by prolonged digestion with urea appears to

be 8~-substituted FAD, judging from the hypsochromic shift of its second ab-

sorption 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 conclu-

sive 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.


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

X-H2C.. ~ .N. N / 0 "-1~ 8 " y ,o "['-" 1 ~ Y

0

Fig. i.

_ N.--~ N NHRI I

I X = I I CH2C H COR2 SD-FLAVIN

N H R I I

2 =-S-CH2CH 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 iso-

lated and purified, and flavin obtained by urea treatment of the flavocyto-

chrome as previously described (3). Proteolytic digestion was performed under

N 2 with 0.i mg each of crystallized trypsin and chymotrypsin per mg of cyto-

chrome 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 Flori-

sil, 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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Vol. 46, No. 3, 1 9 7 2 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

acetic acid-l% pyridine) was run at 50 V/cm. Fluorescence spectra were re-

corded with a Hitachi-Perkin Elmer Model MPF-3 spectrofluorometer equipped

with a spectral correction accessory. 8-Carboxy-riboflavin and 8-a-sulfonyl-

riboflavin were synthesized according to Kenney and Walker (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, the flavin was sub-

jected to mild acid hydrolysis and acid phosphatase digestion to cleave the

I

lOG

Fig. 2. ESR spectrum of the cation radical of Chromatium flavin. The flavin was released by urea treatment, purified, and degraded to the riboflavin level

.... ~u ~.+3 we reco ded (3); 20 nmoles in 6 N HCI reducea wiL~, ~l re r at room temp. with a Varian E-9 spectrometer at 9.1 GHz resonance frequency, 40 mW power, 0.25 G modulation amplitude, i00 KHz modulation frequency, i sec time constant, and 8 min scanning time.

pyrophosphate linkage and remove the 5'-phosphate residue (3). The ESR spec-

trum is essentially identical both in signal width (37 G) and hyperfine struc-

ture (17 lines, 2.3 G spacing) with that of cysteinyl-8a-riboflavin, the fla-

vin component of MAO (6) and differs characteristically from the ESR spectra

of both normal flavins and of histidyl-8~-riboflavin (i0).

For reasons detailed in previous paper s (i0, ii) this type of character-

istic alteration of the ESR spectrum of riboflavin constitutes strong evidence

that the substitution is indeed in the 8a position. 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 (6), the product of trypsin-

chymotrypsin digestion shows a hypsochromic 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 (Fig. 3 and Table II).

This shift in the maximum and the enhancement of fluorescence on oxidation in-

dicate an electron-donating substituent at 8e in the Chromatium flavin. The

possibility that, as in MAO, sulfur is the substituent 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, as given in Methods, the homogeneous product ob-

tained after electrophoresis gave positive tests for reduced S in the I2-azide

and chloroplatinic acid tests.

~j

8 0

6 0

4.0

2 0

I ; I I I

448

352 I [ 3 6 5

f f I I I 3 0 0 4-00 5 0 0

WAVELENGTH (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 conditions

have been compared (Table I). 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, in treatments expected to yield a di-

nucleotide, 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 of an equivalent amount of

riboflavin or FMN, when concentrations are normalized from absorbance at 450

nm (Tables I and II). 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 oxi-

dizing agents as well as mercurials (2). In addition, direct oxfdation of en-

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

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

possibilities have been considered: a cysteine -SH may be joined to (a) an

8e-thioflavin (disulfide), (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 fla-

vin 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

0.i M KOH, 60 ° , 20 min

Same after performic acid oxidation

4 M NH20H , pH 6.1, 60 ° , 20 min

2 mM DTT, pH 7, 37 ° , 8 hr

4 mM dithionite, pH 6.9, 38 ° , 2 hr

Trypsin-chymotrypsin, N2,

2 mM DTT (cf. METHODS) b)

Same after hydrolysis in N HCI

Same after performic acid oxidation

%

80

34

34

0

0

0

7

13

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 oxi-

dized to the sulfone without being released from the peptide under these con-

ditions (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

Criterion

Result

Chromatium flavin a) 8-Carboxy-riboflavin

Fluorescence excitation

spectrum, Ima x at pH 6.0 at pH 2.0

Fluorescence yield, % of

riboflavin

ESR spectrum b)

Electrophoretic mobility c) at pH 1.6

pH 3.4

PKad)

value in TLC e)

448, 368 nm 448, 367 nm 448, 352 nm 448, 352 nm

81 84

17 lines, 2.3 G spacing 17 lines, 2.3 G spacing

+0.2 +0.2 +i.i +I.i

2.5 2.5

0.42 0.42

a) b) c) 1 to o After dephosphorylation, d~ In 6 N HCI. The migration of FMN re ative rib - 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 ex- periment and Mr. R. Seng for skilled assistance.

REFERENCES

1. R.G. Bartsch, Fed. Proc., 20, 43 (1961). 2. R.G. Bartsch, T. E. Meyer and A. B. Robinson, in "Structure and Func-

tion of Cytochromes," K. Okunuki, M. D. Kamen, and I. Sekuzu, eds., University Park Press, Baltimore, Md., 1968, p. 443.

3. R. Hendriks and J. R. Cronin, Biochem. Biophys. Res. Communs., 44, 313 (1971).

4. W.H. Walker and T. P. Singer, J. Biol. Chem., 245, 4224 (1970). 5. W.H. Walker, T. P. Singer, S. Ghisla, and P. Hemmerich, Eur. J. Biochem.,

inpress. 6. W.H. Walker, E. B. Kearney, R. Seng, and T. P. Singer, BiOchem. Biophys.

Res. Communs., 44, 287 (1971). 7. W.H. Walker, E. B. Kearney, R. Seng, and T. P. Singer, Eur. J. Biochem.,

in press. 8. J.R. Kimmel, G. K. Kato, A. C. M. Paiva, and E. L. Smith, J. Biol. Chem.,

237, 2525 (1962). 9. W.C. Kenney and W. H. Walker, FEBS Letters, in press.

i0. 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.

ii. 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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Documents

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