THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 247, No. 9, Issue of May 10, pp. 2768-2775, 1972

Printed in U.S.A.

Tryptic Conversion of Cytochrome b5 Reductase to an

Active Derivative Containing Two Peptide Chains*

(Received for publication, January 13, 1972)

PHILIPP STRITTMATTER, RONALD E. BARRY, AND DORIS CORCORAN

From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 060.%?

SUMMARY

Cytochrome bS reductase has been converted to an active, lower molecular weight flavoprotein derivative by incubation with high concentrations of trypsin. A total of 47 amino acid residues is removed from the single polypeptide chain of this enzyme to form a flavoprotein of 28,400 daltons composed of two peptide chains and 1 molecule of FAD. Noncovalent interactions between the smaller peptide of 75 amino acid res- idues and the larger, 158 to 170 amino acid segment provide a conformation which binds flavin and retains the structural features essential for NADH cytochrome bS reductase activ- ity. The maximum velocity is 65 % of that of the unmodified enzyme. Procedures have been described for the isolation of the separate peptide fragments, and recombination experi- ments show that both peptides are necessary for refolding to form a catalytically active flavoprotein. The larger peptide fragment includes both tryptophanyl residues present in the unmodified enzyme, and indirect evidence suggests that this larger peptide fragment may also contain the sulfhydryl group essential for pyridine nucleotide interaction and structures di- rectly related to flavin binding.

Several structural features of cytochrome bg reductase (EC 1.6.2.2) involved in the precise sequence of coenzyme and sub- strate interactions during the stereospecific oxidation of NADH and reduction of cytochrome bs (1, 2) have been recognized by reversible denaturation studies (3-6). Thus, refolding of the single polypeptide chain of the enzyme at pH 8.5, after exposure to pH 1.5 at 2”, progresses through several protein conforma- tions which can be distinguished by the tryptophan fluorescence yield, the reactivity of five sulfhydryl groups, the quenching of NADH and flavin fluorescence, and the ability of the protein to bind flavin to yield the absorption spectrum and catalytic prop- erties of the native enzyme (4). Whereas the intermediate conformations formed during refolding following acid denatura- tion can only be stabilized by mercurials, two stable forms of the reductase, the active holoenzyme and an inactive, less com- pact flavoprotein species, have been obtained at pH 10.7 to 11.7 (6). In the latter case, the transition from, the holoenzyme to the “swollen” conformation clearly involves the exposure of 1

* This investigation was supported by Research Grant GM- 15924 from the United States Public Health Service.

cysteinyl and 1 tyrosyl residue to the medium, and destroys the pyridine nucleotide-binding site. Moreover, the transitions be- tween the two reductase structures, characterized by a marked hysteresis during a cycle of inactive enzyme formation and re- generation of active enzyme, are dependent upon the state of ionization of basic amino acid residues and the reversible ex- posure of the buried tyrosyl and sulfhydryl residues. Of par- ticular significance for the experiments described here, was the observation that the “swollen” enzyme form, in contrast to the active enzyme, is attacked by low concentrations of proteolytic enzymes. Incubation of the inactive enzyme form with trypsin produces a stable flavin-binding peptide of approximately 10,000 molecular weight.

The objective of the present study was to determine whether or not the entire primary structure of the single peptide chain of cytochrome b5 reductase is required for a stable, active en- zyme. Incubation of reductase with high concentrations of trypsin produces an active derivative (reductase-T), which is composed of two peptide segments containing a total of 245 amino acid residues rather than the 292 residues of the native enzyme. Noncovalent interactions between the smaller pep- tide of 75 residues and the larger, 158 to 170 amino acid segment provide a conformation which is relatively stable, binds flavin, and retains the essential features required for the NADH cyto- chrome bs reductase and ferricyanide reductase activities of the unmodified enzyme (7-9). This altered flavoprotein species undergoes reversible denaturation and can be separated into the component peptides by gel filtration under various denaturing conditions.

EXPERIMENTAL PROCEDURE

Ferricyanide and cytochrome bs reductase activities of cyto- chrome bS reductase preparations and derivatives were meas- ured at 25”, and maximum velocities and K, values were deter- mined from Lineweaver-Burk plots as described previously (5, 10, 11). Absorption spectra were recorded at 2” with a Bausch and Lomb Spectronic 505 recording spectrophotometer, which has an expanded scale and a 5-A band width from 200 to 700 nm. The extent of enzyme flavin reduction with NADH was determined by measuring the decrease in absorbance at 461 nm, assuming that complete reduction of the flavin results in 8576 bleaching at this wave length (3).

Fluorescence spectra were measured at 2” with 0.65- or 0.70- ml samples in l-cm square cells with optics arranged to detect fluorescence at 90” to the exciting light. The spectrofluo- rometer, constructed of Bausch and Lomb component optics and

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a sensitive detector system, has been described (6). A mono- chromator with a 7.4 nm per mm of dispersion was used for excitation wave lengths with an exit slit of 1.0 mm. The mono- chromator employed for emission detection has a 1.6 nm per mm of dispersion, and a 5-mm slit width was employed.

Reactive sulfhydryl groups of various enzyme preparations were determined by the method of Boyer (12) with mercuri- benzoate in Tris-acetate buffer, pH 8.5, at 2”. Mersalyl bind- ing to the reductase preparations was determined by the quench- ing of the fluorescence of this mercurial when it binds to the protein as described previously (4). Estimation of the trypto- phan and tyrosine content of proteins and peptides was based upon the ultraviolet spectra at neutral and alkaline pH values according to the procedures and extinction values of Beaven and Holiday (13).

Sedimentation velocity measurements of dilute protein solu- tions were carried out with a Beckman model E analytical ultra- centrifuge equipped with ultraviolet scanning optics. The standard procedure of Schachman (14) was used to calculate sedimentation constants from the equation s = (din r/dt) (l/ ~2). The sedimentation rate, din r/d& was calculated from an unweighted least squares fit of the data. Molecular weights were determined by equilibrium sedimentation (15) with the same scanning optics used to measure protein concentration gradients at equilibrium. Protein and reference buffer solu- tions were placed in a six-place Yphantis cell (15) and brought to equilibrium at 16,000 rpm. The equilibrium data, the par- tial specific volume of 0.737 of the reductase determined pre- viously (16), and the partial specific volume of 0.740 of reduc- tase-T, calculated from the amino acid composition as described by Schachman (14), were then used to calculate molecular weights from the standard equation, M = [2RT/(l - ~p)w”] (din c /dr2) .

The method of Weber and Osborn (17) was used for acryl- amide gel electrophoresis for the determination of polypeptide homogeneity and molecular weight estimations. The gels were 13.5% cross-linked and contained 0.17, sodium dodecyl sulfate and 0.1 y0 mercaptoethanol. Samples were previously incu- bated for 30 min at 25” in 0.04 M dithioerythritol prior to analy- sis to eliminate possible disulfide artifacts, particularly with iso- lated peptides.

Amino acid analysis was performed by the technique of Spa&man, Moore, and Stein (18) as described previously (19). The determination of the NHrterminal amino acid residues utilized the Edman procedure (20) and cysteinyl and methionyl residues were determined as cysteic acid and methionine sulfone after performic acid oxidation as described previously (19).

The preparation of calf liver cytochrome b5 reductase has been described (3, 6). The more recent modified procedure (6)) which utilizes extraction at pH 5.5 in the absence of snake venom, was used to prepare the enzyme for this study.

Preparation and Isolation of Reductase-T-Conversion of re- dudase to reductase-T was achieved by dissolving 16 mg of TCPK-trypsinl in 4.5 ml of 10-G M reductase in 0.1 M Tris-ace- tate buffer, pH 8.1, adding 0.10 ml of 2 M Tris and incubating for 2 hours at 25”. This solution was then cooled to 2” for all subsequent procedures, and placed on a DEAE-A50 Sephadex column (1 x 15 cm) equilibrated with 0.1 M Tris-acetate buffer,

1 TPCK-trypsin refers to trypsin treated with L-l-tosylamido-2- phenylethyl chloromethyl ketone to inhibit contaminant chymo- tryptic activity.

pH 8.1. The column was washed with 50 ml of the same buffer containing 0.05 M sodium chloride to remove trypsin in the elu- ate, and to move the reductase-T, as a bright yellow band, to within 4 cm of the bottom of the column. The upper 10 cm of the gel were then removed, and the reductase-T was subsequently eluted in a volume of approximately 2 ml with Tris-acetate buffer containing 0.5 M sodium chloride, pH 8.1. The reduc- tase-T solution was subjected to ammonium sulfate fractionation (72 g of ammonium sulfate and 0.5 ml of concentrated ammo- nium hydroxide per 100 ml of solution). The yellow precipitate which formed between 62 and 85% saturation was collected by centrifuging at 15,000 x g for 10 min, and dissolved in 0.5 ml of 0.02 M Tris-acetate buffer, pH 8.4. Gel filtration of this flavoprotein solution on a Sephadex G-25 column (1 x 25 cm) equilibrated with 0.01 M Tris-acetate buffer, pH 8.4, yielded a solution of reductase-T representing an 80 to 95yc recovery of the original enzyme in the trypsin-modified form.

The NADH, FAD, mercuribenzoate, and mersalyl were ob- tained from the Sigma Chemical Company. TPCK-trypsin was a product of Worthington Biochemical Corporation. Sephadex gels were obtained from Pharmacia, and Bio-Gel preparations were from Bio-Rad Laboratories.

RESULTS

Tryptic Conversion of Reductase to Reductase-T-A comparison of native reductase and an inactive “swollen” conformation of this flavoprotein (6) indicated that cytochrome b5 reductase is relatively insensitive to trypsin. This observation suggested that high concentrations of trypsin might produce limited, selec- tive cleavages in the single polypeptide chain of the enzyme. Preliminary experiments revealed that incubation of 1 to 100 nM reductase with 10 to 100 nM trypsin in 0.1 M Tris-acetate buffer, pH 8.1, for 1 to 5 hours at 25” caused no release of flavin, did not alter the behavior of the flavoprotein upon gel filtration with Sephadex G-75, and resulted in the loss of 5 to 35% of the catalytic activity. However, the electrophoretic mobility of the tryptic product on cellulose acetate strips was increased, and most significantly acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the original single peptide chain of the reductase was replaced by two peptides of lower molecular weight. The latter observation is illustrated in Fig. IA, which compares the native enzyme and the trypsin-modified enzyme (reductase-T) prepared by the standard procedure described under “Experimental Procedure.” This method re- moves contaminating traces of trypsin and provides reductase-T virtually free of unmodified enzymes in good yields. Such puri- fied preparations were used for subsequent characterization of reductase-T.

Both Peptides T-l and T-2, as well as the flavin are integral, tightly bound components of reductase-T. This is implied by the fact that a single flavoprotein, containing both peptides, is isolated by purification involving DEAE-Sephadex column chromatography, ammonium sulfate fractionation, and gel filtrat#ion. Moreover, the interactions between Peptides T-l and T-2 result in a stable apoprotein preparation. When flavin is removed from reductase-T by the acid charcoal procedure described for aporeductase preparation (4), Peptides T-l and T-2 remain in a stable association from pH 2 to 10 and cannot be separated by gel filtration. As will be described below, only severe denaturing conditions, such as 30% acetic acid, 0.3%

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1 2 FIG. 1. Acrylamide disc gel electrophoresis of reductase, re-

ductase-T, and Peptides T-l and T-2. Each set, designated A and 13, includes gels from a single electrophoresis experiment in sodium dodecyl sulfate buffer as described under “Experimental Proce- dure.” (gel 8).

A, a comparison of reductase (gel 1) and reductase-T B, from left to right, peptides in tubes 28 through 39 from

the Bio-Gel P-10 separation of Peptides T-l and T-2 described in Fig. 4 and the text.

I

I , ^- -.-

- 260 300 340 380 420 460 500 WAVELENGTH (nm)

sodium dodecyl sulfate, or 7 M urea permit separation of the two peptides on Sephadex or Bio-Gel columns.

Catalytic, Spectral, and Fluorescence Properties of Reductase-T- Table I compares the catalytic activities of reductase-T to those of the native enzyme. The maximum velocity, with either electron acceptor, is decreased by approximately 40y0 and the K, values for both the reduced pyridine nucleotide and cyto- chrome b5 are increased approximately 5-fold. Alteration in the protein structure is thus sensitively reflected in modified interactions of both NADH and cytochrome b5 with reductase-T.

The absorption spectrum of reductase-T (Fig. 2, Curve 1) is very similar to that of the unmodified enzyme (Curve 2). Flavin binding is reflected in the red shift in the isoalloxazine absorption bands compared to free FAD (Curve 3) and discernible band splitting, particularly in the 400 to 500 nm wave length region. There are slight differences between the two reductase prepara- tions, largely in the intensity of the 392 nm absorption band, but

I I I I I

TABLE I Catalytic activities of reductase and reductase-T

Preparation

v,EXa

Cytochrome bs Ferricyanide

KC%

NADH cyto- chrome bs

PM

Reductase. . . . . , . . . 30,000 31,000 2.7 20 Reductase-T. . . . . 16,900 21,000 16.0 91

o Values are expressed as micromoles of NADH oxidized per min per pmole of enzyme with either cytochrome bc or ferricyanide as electron acceptor. See “Experimental Procedure” for assay conditions.

-

0.05 d 0 m 6

0 0’ I I 3;o

I 4;o

1 320 WAVELENGTH (nm)

FIG. 2 (left). Absorption spectrum of reductase-T. ductase-T; Curve 8, reductase; Curve 8, FAD.

Allsamples were 5p~ in 0.1 M Tris-acetate buffer, pH 8.1, at 2”. Curve 1, re-

FIG. 3 (right). Fluorescence emission spectra of reductase and reductase-T at 2’. the proteins were in 0.1 M Trisacetate buffer, pH 8.1.

The excitation wave length was 295 nm, and Curve 1, 2.7 NM reductase; Curve 2, 2.7 PM reductase-T.

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these appear in the ultraviolet wave length region as well. Since reductase-T still contains all of the tryptophanyl and tyrosyl residues, with the same absorption spectra in the ultraviolet wave length region as the unmodified enzyme (see below), all of the spectral differences appear to be attributable to significant alterations in the flavin environment during the conversion of reductase to reductase-T. Nevertheless, the quenching of the isoalloxazine fluorescence is complete in reductase-T, as was ob- served previously for the intact reductase (3).

The emission band of the 2 tryptophanyl residues of cyto- chrome bs reductase has a maximum of 338 nm (6). A compari- son of the emission spectra of reductase and reductase-T (Fig. 3, Curves i and d) shows that tryptic modification of the flavopro- tein causes both a red shift to 344 nm and a 32y0 greater quench- ing of the fluorescence of the indole rings. The structural changes in forming reductase-T, which result in alterations in both the catalytic constants and flavin absorption spectrum, thus include a movement of one or both tryptophanyl residues to a more polar environment.

Separation of Peptides T-l and T-d Gel Filtration-Gel filtration with B&Gel P-10 columns equilibrated with 0.3% sodium dode- cyl sulfate in 0.1 M Tris-acetate buffer, pH 8.1, 30% acetic acid, or 7 M urea separates Peptide T-l, Peptide T-2, and flavin. A typical procedure is illustrated in Fig. 1B and Fig. 4 with a Bio- Gel column equilibrated with the detergent solution. The ultraviolet absorbance was monitored at 288 and 278 nm (Fig. 4) to distinguish peptides in the effluent containing both tryptophan and tyrosine or predominantly one of these amino acids. The data are shown from the exclusion volume (tube 27) to tube 40. These tubes contained all ultraviolet absorbing material except the flavin, which was completely included and recovered in

I I I I I / / , /

FRACTION NUMBER

-FIG. 4. Separation of Peptides T-l and T-2 by gel filtration. A 0.65-ml sample of 0.105 IrIM reductase-T in 0.15y0 sodium dodecyl sulfate was placed upon a column (0.9 X 67 cm) of Bio-Gel P-10 equilibrated-with 0.3-y0 sodium dodecyl sulfate. The column was developed at a flow rate of approximately 0.7 ml per hour; 0.5.ml fractions were collected in separate tubes, and monitored for absorbance with 0.3% sodium dodecyl sulfate as a reference blank. All ultraviolet absorbing material, with the exception of free Ravin, appeared in tubes 27 through 40, and the flavin was col- lected in tubes 55 to 57. Curve 1, absorbance at 278 nm; Curve 8, absorbance at 288 nm.

tubes 55 to 57. Acrylamide gel electrophoresis of aliquots of tubes 28 through 39 (Fig. 1B) show that the small amount of residual unmodified peptide of the enzyme appears first, then Peptide T-l and finally the smaller Peptide T-2. Moreover, it is clear from Fig. 4 that Peptide T-l contains both tryptophanyl and tyrosyl residues, whereas Peptide T-2 has no tryptophan. To obtain either pure Peptide T-l or T-2 from this and all other preparations with any one of the three solvent systems, tubes containing only Peptides T-l or T-2 by both the gel electrophore- sis and absorbance criteria were combined.

Tryptophanyl, Tyrosyl, and Suljhydryl Residues o.f Reductase-T and Peptides T-l and T-,%-The millimolar extinction values of aporeductase and aporeductase-T from 240 to 350 nm are vir- tually identical (Fig. 5, Curves 1 and 2). The tryptic modified enzyme therefore has the same tryptophan and tyrosine content as the reductase (2 tryptophan and 8 tyrosine residues) (6) as determined by the spectral and alkaline difference spectrum method of l3eaven and Holiday (13). For both apoenzyme preparations, the protein concentrations were determined from the flavin content of the holoenzyme prior to FAD removal. In the case of Peptides T-l and T-2 (Curves 3 and 4), the calculation of molar concentrations was based upon the fact that both tryptophanyl residues are in Peptide T-l, since all ultraviolet absorbing material from 260 to 300 nm is recovered in the Bio- Gel separation of Peptides T-l and T-2, and tryptophan occurs only in Peptide T-l. The concentration of Peptide T-l was therefore calculated directly from the tryptophan absorbance, and, consequently, from the tryptophan to tyrosine ratio, this peptide must also contain 4 of the 8 tyrosyl residues of reductase- T. The remaining 4 residues of tyrosine must then be in Pep- tide T-2, and, as expected, the spectrum of this peptide indicates that it contains only tyrosine and no tryptophan. Molar con- centrations of Peptide T-2 were calculated by assuming that it contains 4 tyrosyl residues.

The number of sulfhydryl groups in reductase-T and Peptides T-l and T-2 was determined under denaturing conditions with mercuribenzoate (Table II, Lines 2, 5, 7, and 8). Trypsin re- leases 1 of the 5 detectable cysteinyl residues of cytochrome bs reductase, and 3 of the remaining 4 are in Peptide T-l (Line 7)

260 280 300 WAVELENGTH (nm)

I

URVE -I

77-T

. . . . 2 ---_ 3 --- 4

I-,

FIG. 5. Absorption spectra of aporeductase, aporeductase-T, and Peptides T-l and T-2. Spectra were recorded in 0.1 M Tris- acetate buffer, pH 8.1, at 2”. Concentrations of proteins and peptides were determined a,s described in the text. Curve 1, aporeductase; Curve 2, aporeductase-T; Curve S, Peptide T-l; Curve 4, Peptide T-2.

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TABLE II TABLE III Reactivity of sulfhydryl groups in reductase-T and

Peptides T-l and T-2 Amino acid composition of reductase-T and Peptides T-l and T-2

The values for amino acid content are averages from 18- and 48-hour hydrolysates, and cysteine and methionine were de- termined after performic acid oxidation as cysteic acid and methionine sulfone. Tryptophan was determined from the ultraviolet absorbance as described by Beaven and Holiday (13). Reductase values represent an average of 10 analyses, reductase- T, of 5 analyses, and Pel 3ti des T-l and T-2, of 2 analyses.

Preparation Conditiona

Reductase pH 2.0 Reductase pH 8.1, sodium dodecyl sulfate Reductase pH 8.1 Reductase-T pH 2.0 Reductase-T pH 8.1, sodium dodecyl sulfate Reductase-T pH 8.1 Peptide T-l pH 8.1, sodium dodeeyl sulfate Peptide T-2 pH 8.1, sodium dodecyl sulfate Reductase pH 8.1, NADH Reductase-T pH 8.1, NADH

-

-

Mercuri- benzoate

bound

W%Ol‘2S/?id~ *re*aration

5.00 4.05 3.00 4.00 3.95 3.02 3.05 0.92 2.0 2.0

Q All preparations were 5 PM in 0.1 M Tris-acetate buffer, pH 8.1, and 2”. For the low pH measurements, a IO-fold molar excess of mercuribenzoate was added to preparations adjusted to pH 2.0 with 1 N HCl, and the solutions were then quickly brought to pH 8.1 with 2 M Tris to determine the mercuribenzoate bound by the method of Boyer (12). As indicated, some samples con- tained either 0.3yo sodium dodecyl sulfate or a a-fold molar excess of NADH.

and the fourth is found in Peptide T-2 (Line 8). The residue lost during the conversion of reductase to reductase-T is identi- fied as the residue which is exposed to the mercurial only at a low pH (4).

Earlier examination of the unmodified reductase (4, 11) has shown that the sulfhydryl residues can be distinguished by dif- ferences in reactivities. One of the five residues is directly in- volved in NADH binding, three are normally reactive with mercuribenzoate and the two “buried” residues will react with mercurials during specific stages of unfolding and refolding of the protein during reversible denaturation (4). Similar attempts to characterize the 4 sulfhydryl residues in reducatse-T are sum- marized in Table II. Lines 3 and 6 indicate that both forms of the enzyme contain three reactive sulfhydryl groups, and Lines 9 and 10 show that in each case, one of these residues is protected by NADH. The cysteinyl residues, which are normally reactive in the reductase and include the residue essential for substrate interaction, therefore appear to be retained in reductase-T.

Amino Acid Composition and Molecular Weights of Reductase-T

and Peptides T-l and T-2-Column 3 of Table III shows that the formation of reductase-T from reductase results in the loss of 47 amino acid residues, representing a decrease in molecular weight of 5600. Because very large amounts of trypsin are used in the preparation of reductase-T, peptide fragments could not be recovered, and the accuracy of estimating the loss of amino acids was limited to the determinations of the differences in amino acid composition of the flavoproteins. The two peptides of reductase-T account for all of the amino acids of reductase-T wit,hin the precision of the amino acid analysis. The sum of residues in the two peptides is within 57, of the composition determined for reductase-T. Because the amino acid analyses of Peptide T-l were somewhat more variable and yielded mo- lecular weight values slightly lower than expected from molecular weight determinations based upon gel electrophoresis (see below),

Amino acid

Lysine. ................ Histidine. .............. Arginine ............... Aspartic acid. .......... Threonine. ............. Serine .................. Glutamic acid. ......... Proline. ................ Glycine. ............... Alanine. ............... Cysteine. .............. Valine. ................. Methionine ............. Isoleucine .............. Leucine ................ Tyrosine ............... Phenylalanine. ......... Tryptophan .............

Total. .................

Reductase

20 16 12 5 9 7 3 3

16 12 5 5 28 25 15 9 14 11 6 4 13 12 6 4 29 23 19 4 29 22 16 8 20 19 13 4 15 14 9 2 6 4 3 1

16 15 9 5 8 8 7 1

19 17 9 6 27 20 14 7 8 8 4 4

13 10 6 3 2 2 2 0

292 245 158 75 -

TABLE IV Molecular weights of reductase-T and Peptides T-1 and T-2

I Molecular weighta

Peptide T-2

Preparation Amino acid

analysis

Reductase 34,006 Reductase-T 28,400 Peptide T-l 17,806 Peptide T-2 8,674

Equilibrium Gel sedimentation electrophoresis

35,400 (6) 34,000 30,900

21,900 8,400

a See “Experimental Procedure” for methods. The protein molecular weight values by amino acid analysis and sedimenta- tion include the Aavin; other values do not.

the amino acid composition of Peptide T-l is less certain. There- fore, the amino acid composition of Peptide T-l is reported at 158 to 170 residues, the values for the direct analysis and the difference between reductase-T and Peptide T-2, respectively. Analysis for cysteic acid, following performic acid oxidation, suggest that unmodified reductase may contain a total of 6 rather than the 5 cysteinyl residues detected by mercuribenzoate titrations. In view of the difficulties in obtaining extremely accurate cysteine values, however, the occurrence of a sixth, totally unreactive sulfhydryl group in the reductase is not cer- tain. In any event, reductase-T contains only 4 residues of this amino acid.

Molecular weight determinations by several methods are

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0 I.-.-.,----L23 340 380 420 460 500

WAVELENGTH (nm)

FIG. 6. Trypsin modification of flavin binding to mercurial complexes of reductase-T at 2”. Samples of 4.G PM reductase-T, in the presence or absence of a IO-fold molar excess of mercurial, were incubated with 1.2 PM trypsin in 0.1 M Tris-acetate buffer, pH 8.4, for 1 hour. Curve 1, reductase-T without mercurial; Curve 2, reductase-T after the determination of the spectrum shown in Curve 1 and addition of excess NADH; Curve S, reductase- T incubated with trypsin in the presence of mersalgl; Cuurve 4, re- ductase-T incubated with trypsin in the presence of mercuriben- zoate.

summarized in Table IV. The agreement between these analyses is good considering the experimental variation normally encoun- tered with the procedures employed. The greatest difference is 20yc in the molecular weight of Peptide T-l and this compares data from amino acid analysis to results of acrylamide gel elec- trophoresis. Deviations of similar magnitude have been ob- tained wit’h other proteins in applying the gel electrophoresis method (17).

Properties of Reductase-T-The conversion of reductase to reductase-T decreases the stability of t’he flavoprotein. This is reflect.ed in flavin dissociation when reductase-T is incubated at 35” in 0.1 M Tris-acetate buffer, pH 8.1, for 3 hours, or at 27.5” in the same buffer in the presence of mersalyl or 4 M sodium bro- mide for shorter periods of time. These conditions do not effect the spectrum or activity of the unmodified enzyme. Moreover, the preparation of a stable, ‘Lswollen” derivative of reduct,ase at pH 10.5 to 11 (6) cannot be repeated wit.h reductase-T; instead, flavin dissociation occurs.

Reversible modification of reductase-T occurs, on t,he other hand, under conditions wherein the original reductase is com- plet’ely stable. Bot’h forms of t,he enzyme are st’able in 2 M sodium bromide in 0.1 M Tris-acetate buffer, pH 8.1, and 2”. The addition of 4 eq of mersalyl to the protein solutions does not effect the absorption spectrum of reductase, but the spectrum of reductase-T changes from that of the holoenzyme to that of free FM>. This flavin di&ociation from reductase-T is rapidly and c&pletely reversed by the addition of a 5-fold molar excess of dit’hioerythritol. Two moles of mersalyl or a lo-fold molar esces5 of mercuribenzoate do not effect the Aavin binding.

Further characterization of concomitant alterations in the peptide structure during such Aavin dissociation was deferred, and, inst’ead, an examination of t’he effects of mercurials on reductaqe-T under milder conditions, i.e. in the absence of salt, was initiated. In 0.1 M Tris-acetate buffer, pH 8.1, the spectrum and sediment’ation constant of reductase-T are unaffected by a lo-fold molar excess of either mersalyl or mercuribeuzoate. JIorc subt.le alterations of the modified flavoprotein cont,aining

1 2 3 4 5 1 2 FIG. 7. Acrylamide disc gel electrophoresis of trypsin-modified

mercurial complexes of reductase-T. Each set, A and R, inclltdes gels from a single electrophoresis experiment in sodillm dodecyl sulfat,e buffer as described under “Experimental Procedure.” 11, samples of 5.5 PM reduct,ase-T, in the presence or absence of GO PM mercuribenzoate, were incubated with 1.2 PM trypsin in 0.01 M Tris-acetate bluffer, pH 8.4, at 2”. Sodium dodecyl sulfate was added to stop proteolysis after incubation for the intervals in- dicated below. 1, reductase-T incubated for 2 hours; d to 5, re- ductase-T ~111s mercuribenzoate after 15, 30, 60, and 120 min of incubation with trypsin. II?, 1, a sample of 5.5 PM reductase-T was first reduced by the addition of excess NADH (60 PM), and subse- quently incubated for 120 min with mercuribenzoate and trypsin as described for gel 5 of -4 ; 6, a sample of untrcatcd rcduct,asc-T. The electrophoreses were carried out for 3 and 5 hours for A and H, respectively; hence, the differences in the positions of Peptides T-l and T-2.

mercurials were revealed by marked increases in the sensitivity of mercurial complexes of the protein to low concentrations of trypsin at 2” (Fig. 6). In the absence of mercurial, incubation with trypsin has no effect upon the spectrum of reductase-T (Curve 1) or its complete reduction by N,IDH (Curve 2). In contrast, a low concentration of trypsin converts both the mer- salyl-enzyme complex (Curve S) and the mercuribenzoate deriva- tive (Curve 4) into modified flavoprotein species in which the flavin binding is altered, and reduction by NADH in the presence of excess dithioerythritol is irreversibly lost. Nevertheless, the flavin remains tightly bound, since inactive enzyme derivatives which elute as single flavoprotein species upon gel filtration on either Sephadex G-25 or G-75 columns show no flavin fluorescence and retain all of the FAD after prolonged dialysis at 2”.

Acr)-lamide gel electrophoresis clearly shows that the tryptic attack upon reductase-T complexes with mercurials is restricted to Peptide T-l. Fig. 76 shows that trypsin converts Peptide

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2774 Trypsin-modified Cytochrome bg Reductase Vol. 247, No. 9

TABLE V

Reversible denaturation of reductase-T

Preparation Condition for Time for denaturation

I I

recombi- Recombi- nation0 nationa

Reductase-T 307, acetic acid Reductase-T 30% acetic acid Reductase-T 7 M urea Reductase-T 0.1 N NaOH Peptide T-l 30% acetic acid Peptide T-2 307, acetic acid Peptides T-l + T-2 30% acetic acid Peptides T-l + T-2 30$& acetic acid

hrs % 8 55

48 71 48 61 12 68 48 0 48 0

8 35 48 45

Q For recombination at 2”, 0.1 ml of 0.1 mM protein or peptide samples were either dialyzed or passed through Sephadex G-25 columns to change from the denaturing solvent to 0.1 N acetic acid. One equivalent of FAD was then added, and the solution was adjusted to pH 8.4 by the addition of 2 M Tris.

b Recombination was estimated by measuring the extent of flavin reduction by NADH as the peptides refold to form active flavoprotein as described previously (4).

T-l to a smaller fragment, whereas Peptide T-2 remains unal- tered. The small peptide or peptides removed by this proteolytic attack do not bind stain sufficiently to detect them clearly in these gels, and alternate characterization has not yet been at- tempted. Of more immediate interest is the observation that tryptic attack on the mercurial complexes of reductase-T can be completely blocked by prior addition of NADH to the flavopro- tein (Fig. 7B). Since the data in Table II show that NADH reaction with reductase-T protects one of the three reactive sulfhydryl groups from mercurials, the protection from tryptic attack suggests that conformational changes associated with mercurial binding to the residue essential for pyridine nucleotide interactions are critical for this limited proteolytic attack. Moreover, the fact that only Peptide T-l becomes labile repre- sents indirect evidence that the essential sulfhydryl group may be one of the 3 cysteinyl residues in Peptide T-l.

Reversible Denaturation of Reductase-T-Reductase-T can be reversibly denatured under conditions which result in complete flavin dissociation and separation of Peptides T-l from T-2, i.e. by incubation in either 30% acetic acid or 7 M urea (Table V). In 0.1 M alkali, flavin dissociation also occurs readily, but it has not been determined that the two peptides are dissociated. Re- combination to form active enzyme is relatively slow after incu- bation under any one of the three conditions. This differs from the rapid recombination following flavin dissociation induced by sodium bromide and mersalyl, and presumably arises from the slow, proper reassociation of Peptides T-l and T-2. Some ir- reversible denaturation does accompany the cycle of denaturation and refolding, so that only 65 to 75% of the active reductase-T is recovered. The last three lines of Table V indicate that both peptides are required for the active enzyme conformation. Neither Peptide T-l or Peptide T-2 alone will bind FAD to yield an enzymatically active flavopeptide under conditions which result in a slow recombination of the two peptides and flavin to yield 45y0 reductase-T.

DISCUSSION

The early studies of Richards (21) with pancreatic ribonuclease and a series of papers by Anfinsen et al. on a bacterial nuclease

(22-25) have demonstrated that breaks and deletions in the primary structure of this class of hydrolytic enzymes, composed of single polypeptide chains of approximately 120 to 150 amino acid residues, can produce active complexes of peptide fragments. Moreover, the latter workers have shown that fragments of the bacterial nuclease form active complexes, involving interactions of overlapping, redundant regions of the peptide fragments, and have utilized solid phase synthesis of analogues of these fragments to probe essential features of these interact.ions (25). It appears, from the initial experiments reported here, that a similar detailed examination of the structural basis of the catalytic activity of the flavoprotein, cytochrome b5 reductase, is feasible. This flavoprotein is also a single polypeptide, but it is twice as large with 292 amino acid residues, and it fortunately contains distinct binding sites for flavin (3), reduced pyridine nucleotide (26), and a heme protein (1) in a single catalytic site. It is therefore possi- ble in this case to ident.ify specific areas and interactions involved in catalysis, and to recognize their dependence upon particular aspects of the polypeptide structure.

The restricted tryptic cleavage to form reductase-T is de- pendent upon the stabilization of the compact flavin-polypeptide conformation of the active holoenzyme. This form of the en- zyme, which is resistant to extensive tryptic attack at the 21 lysyl and 16 arginyl sites in the primary structure, contrasts markedly with the rapid irreversible destruction of the apopro- tein by extremely low concentrat#ions of t,rypsin (6), or conversion of an alternate, “swollen” conformational state of reductase to an inactive flavopeptide of approximately 10,000 daltons (6). In the presence of a nearly equal amount of trypsin, limited peptide bond cleavage in the active enzyme releases only 167C of the amino acids, and produces the active flavoprotein species with one 75 and one 158 to 170 residue peptide fragment. The positions of these two tryptic peptides, within the primary struc- ture of the original polypeptide, as well as the amino acid residues which are lost, have not yet been determined. However, the rapid methods for the isolation of pure Peptides T-l and T-2, and the flavopeptide produced from the “swollen” form of the enzyme (B), provide a significant simplification in present efforts to determine the sequence of the entire polypeptide chain of 292 residues.

The two large peptide fragments and FAD retain the struc- tures critical for the formation of substrate and coenzyme com- plexes, and the sequence of interactions between NADH, FAD, and cytochrome b5 involved in the over-all oxidation-reduction reaction (1). The unmodLied enzyme has a remarkably high turnover number with either ferricyanide or cytochrome bS as

electron acceptor, and this activity is decreased only approxi- mately 40% in reductase-T. In spite of the fact that the cata- lytic efficiency of reductase-T remains high, the significant de- crease in maximum velocity, increases in the K, values for sub- strates and the aherations in the spectrum of the bound FAD indicate that reductase-T does not retain the precise structural features of the unmodified enzyme that optimize substrate, coenzyme, and protein interactions.

An alteration in the environment of one or both tryptophanyl residues accompanies these modifications of the catalytic ac- tivity. All of the 8 tyrosyl and 2 tryptophanyl residues appear in Peptides T-l and T-2, but the red shift in the fluorescence emission spectrum of the indole rings is consistent with the as- sumption that one or both of these normally “buried” residues (6) are now more exposed to the polar buffer. Both forms of the enzyme, on the other hand, contain three reactive sulfhydryl

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Issue of May 10, 1972 P. Xtrittmatter, R. E. Barry, and D. Corcoran 2775

residues, including one which is involved in NADH binding. With respect to this amino acid, reductase-T differs from reduc- tase in that the two peptide fragments include only one addi- tional unreactive or “buried” cysteinyl residue.

The effects of mercurials binding to the reactive sulfhydryl groups of reductase-T, in the presence and absence of NADH, provide some further insight into the structural functions of the two peptide fragments. When the 3 exposed residues are con- verted to either the mersalyl or the mercuribenzoate complexes, Peptide T-l becomes selectively labile to very limited proteolytic attack by low concentrations of trypsin at 2”. By adding NADH to form the reduced enzyme-NAD complex (l), the es- sential sulfhydryl is protected from mercurials, and tryptic cleavage of Peptide T-l is blocked. Since Peptide T-l contains three of the four sulfhydryl groups in reductase-T, at least two of the three reactive sulfhydryl groups are in this larger peptide fragment which becomes sensitive to trypsin. The most direct, but not the only, interpretation of these data is that the reactive sulfhydryl groups in Peptide T-l (either two or three) include the 1 residue which is essential for NADH interaction, and that mercurial binding to this mercapto group provides the structural distortion that permits the limited tryptic attack on Peptide T-l. This hypothesis is being tested further by experiments with [14C]N-ethylmaleimide to determine by covalent labeling if all of the reactive sulfhydryl groups appear in Peptide T-l, since then, the essential sulfhydryl residue and the distortion of the flavoprotein by mercurial binding would necessarily be related directly to the larger peptide fragment.

The data on the reactive sulfhydryl groups also suggest that both pyridine nucleotide and flavin binding involve interactions with Peptide T-l. NADH interaction with the enzyme is lost, and the flavin binding is altered by the controlled tryptic modifi- cation of Peptide T-l. Therefore, this larger peptide segment, which interestingly contains both indole rings of the holoen- zyme, is implicated in interactions required for catalysis by the enzyme. Nevertheless, it is clear from the recombination studies that both Peptides T-l and T-2 are required to produce a protein conformation that permits these interactions.

The salt- and mercurial-induced flavin dissociation, which is rapidly reversible, provides a method for preparing undenatured aporeductase-T. Such preparations, an improved procedure for more complete recombination of isolated Peptides T-l and T-2

with FAD, and systematic chemical and proteolytic modifica- tion of the isolated peptides and reductase-T are now being used to attempt to define the contribution of each peptide to the stability of the enzyme conformation more precisely, and, per- haps, to identify an active flavoprotein with an even simpler total primary structure.

A&nowledgments-We are indebted to Dr. Eugene Wampler for the ultracentrifuge experiments and to Dr. Juris 0~01s and Mr. John Bunch for the amino acid analyses.

1. STRITTMATTER, P. (1965) J. Biol. Chem. 240, 4481 2. STRITTMATTER, P. (1965) Fed. Proc. 24, 1156 3. STRITTMATTER, P. (1961) J. Biol. Chem. 236, 2329-2335 4. STRITTMATTER, P. (1967) J. Biol. Chem. 242, 4630-4636 5. LOVERDE, A., AND STRITTMATTER, P. (1968)J. Biol. Chem.243,

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Philipp Strittmatter, Ronald E. Barry and Doris CorcoranContaining Two Peptide Chains

Reductase to an Active Derivative5bTryptic Conversion of Cytochrome

1972, 247:2768-2775.J. Biol. Chem. 

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