THE JOURNAL OF BIOLOGICAL CHE~II~TRY Vol. 246, No. 10, Issue of May 25, pp. 3367-3377, 1971

Printed in U.S.A.

Sulfheme Proteins

I. OPTICAL AND MAGNETIC PROPERTIES OF SULE’MYOGLORIN AKD ITS DERIVATIVES*

(Received for publication, November 2, 1970)

JAY A. BERZOFSKY,$ J. PEISACH,~ AXD W. E. BLUMBERG

From the Departments of Molecular Biology and Pharmacology, Albert Einstein College OJ” dledicine, Yeshiva University, Bronx, New York 1046i, and Bell Telephone Laboratories, Inc., Murray Hill, New Jersey 07#,$

SUMMARY

Ferrous sulfmyoglobin was prepared from the higher oxi- dation state of myoglobin (Mb’“) by reaction at pH 8 with a IS-fold molar excess of (NH&S. The purity (>90%) was greater than any previously reported.

The electron paramagnetic resonance (EPR) of the ferric protein at low pH resembles that of ferric myoglobin, where the heme iron is high spin and is in a nearly’ axial environ- ment. At high pH, the heme iron gives a low spin EPR signal, attributed to the hydroxide derivative, which is dis- tinct from that of ferric myoglobin hydroxide.

Acid-base titration of ferric sulfmyoglobin in the visible and near infrared gives a pK of 8.45 Z!I 0.05 while similar titration of ferric myoglobin yields a pK of 8.9 to 9.0. A computer analysis of the titration data for both compounds confirmed the pK for ferric sulfmyoglobin and, coupled with EPR data, has been used to determine the purity of the preparation.

Evidence is presented that the small amount of impurity in the ferrous sulfmyoglobin preparation is present as oxy- myoglobin. A computed spectrum of pure ferrous sulfmyo- globin is given, and has been used to re-evaluate the purity of preparations described in previous publications.

Fluoride binding to ferric sulfmyoglobin was established by EPR. The low spin ferric cyanide and azide derivatives were studied by optical and EPR spectroscopy. An analysis of the EPR spectra of low spin derivatives of ferric sulfmyo- globin leads us to believe that the electronic distribution at the heme is different from that of ferric myoglobin and, com- bined with an analysis of optical properties, supports the hypothesis of a structure in which the elements of HzS add across a /3-b double bond of a pyrrole and thereby disrupt the porphyrin conjugation and form a chlorin type structure.

* The portion of this investigation carried out, at the Albert Einstein College of Medicine was supported in part by United States Public Health Service Research Grant HE-13399 to J. Peisach from the Heart and Lung Institute. This is Communica- tion 216 from the Joan and Lester Bvnet Institute of Molecular Biology.

$: Predoctoral candidate of the Medical Scientist Training Pro- gram, supported by Grant 5T5-GAI-1674 from the United States PubIic Health Service.

§ Recipient of Public Health Service Research Career Develop- ment Award I-K3-GM-31,156 from I he National Institute of Gen- eral Medical Sciences.

Sulfhemoglobin is found in a number of pathological condi-

tions, particularly those in which a sulfide source is present (1) or in which there is blood poisonin g by certain reducing agents (2, 3). In the latter case, the reducing agents serve as catalysts, as shown only recently by Nichol et al. (4), while the sulfide source is endogenous, probably H,S from intestinal bacteria. Sulfhemoglobin is of practical medical iilterest as one of the main forms of nonfunctional hemoglobin found in the red cell.

The sulfheme proteins represent a class of compounds formed by analogous reactions of various protoheme proteins and H$. These include Sulf-Hb’ (5-9) and sulf-Mb (8, 10, 11) and sulf- catalase (10). Hoppe-Seyler (5), in 1866, was the first to ob- serve a green product formed from the reaction of HbOz and HtS. He called this product “Schwefelmethamoglobin” or sulfhemoglobin.

In 1933, Keilin (6) reported that sulfhemoglobin is formed irreversibly from Hb and H8 only in the presence of 02, that it is distinct from ferric Hb sulfide, in which sulfide is thought to be the distal ligand to the iron, and that the characteristic 618 rnp spectral band for sulfhemoglobin probably belongs to the ferrous form since it is bleached by K,Fe(CE),.

The spectrophotometric analysis of the ferrous protein by Drabkin and Austin (7) 2 years Iater yielded computed extinc- tion coefficients which became the primary standard for many later workers. However, a major difficulty in all early studies was the very low purity of the preparations.

Aware of the effects of reducing agents reported in 1907 by Clarke and Hurtley (la), Michel (2, 8, 13) prepared sulfhemo- globin by a new method, namely the successive addition of dithionite, sulfide, and a peroxide source. By chemical analysis, Michel demonstrated 1 additional sulfur atom per heme, but he ruled out the binding as distal ligand to the heme since CO bound there. He also prepared analogous spectral species from Mb and from hematoheme-substituted hemoglobin, which lacks vinyl groups. Most later workers took advantage of Michel’s observation and used the simpler protein, Illb, for their studies.

1 The abbreviations used are Hb, hemoglobin and Mb, myo- globin, and these are preceded by “sulf” for the corresponding sulfheme compound or followed by a ligand (or both), e.g. MbOz for oxymyoglobin or ferric MbCN for ferric myoglobin cyanide. Roman numeral superscripts denote the total oxidation state of the compound, on a scale where deoxy-Mb is Mb”, ferric Mb is Mbrrr, and the product of the reaction of Mbrrr and HzOz is Mbrv (14). EPR is electron paramagnetic resonance.

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3368 Xulfheme Proteins I: 8dfm~oglobin Vol. 246, No. 10

In 1961, Nicholls (10) synthesized sulf-Mb from the higher oxidation state derivative of Mb, Mb’v, the product of H202 and ferric Mb (14). He showed that the addition of 1 mole of H2S per heme produced ferric sulf-Mb, which could then be reduced to the ferrous compound by excess HZS. He also observed that the ligands, OH-, CN-, I?, and N3, produced ferric sulf-Mb derivatives with optical spectra shifted 50 to 90 rnp to the red of those of the corresponding ferric Mb derivatives.

In 1967, Morel& Chang, and Clezy (11) modified Nicholls’ synthesis to give a slightly better yield of ferrous xulf-Mb, as judged optically. From the resemblance of ferric and ferrous sulf-PIIh optical spectra to those of the iron mesochloringlobin Compounds, in which one of the pyrrole p-/3 double bonds of the heme is saturated, they suggested that a sulfur atom is added across such a double bond to form an episulfide bridge. How- ever, their attempt to demonstrate binding of 35S to the heme of sulf-Mb n-as inconclusive, since significantly more than 1 g atom was bound per mole of protein, and, of this, a large percentage did not follow the hemin when it was extracted from the protein. In the present study, a modification of the methods of Nicholls and of Morel1 et al. has led us to a preparation of sulf-Mb which we show to be of greater purity (at least 90%) than any previ- ously obtained. The use of EPR has enabled a more direct method for assay of purity than had been available to others, as well as providing considerably more‘ information about the electronic structure near the heme than can be obtained from optical spectroscopy alone. This new preparation has allowed the spectrum of pure ferrous sulf-Mb to be computed, and from t,his spectrum the purity of previous preparations to be assessed from published spectra. A computer analysis of a pH t,itration of the ferric s&-Mb has provided furt,her estimates of purity, as well as an accurate pK and computed spectra of the pure high >rnd low pH species.

EXPERIMENTAL PROCEDURE

Ikfaterials

Sperm whale oxympoglobin was prepared according to the method of Hugli and Gurd (15). The protein was oxidized with excess K,Fe(CN)G followed by passage through Rio-Gel P-6 to remove excess reagent. The ferric Mb was concentrated in a Sartorius collodion bag by ultrafiltration and stored frozen at a concentration of about 3 mM. It was free of low spin material (less than 0.1 s) by EPR criteria (16). Hydrogen peroxide was the Fisher certified reagent, ACS, “30%” grade. Its concentra- tion, determined by titration with KI and standard thiosulfate, was 29.8y0 (8.73 or) and was st,able for periods of at least 6 months at 4”. Ammonium sulfide was t,he Fisher certified reagent, “light” solut’ion. Jt was titrated by the method of Kolthoff and Sandell (17) by osidat’ion of S= to SO,= with a known excess of NaOCl which was standardized against thiosulfate. The excess OCl- was titrated with KI and standard thiosulfate. This method yielded 4.4 and 4.2 M (NHJZS in two separate experi- ments. Crystalline bovine liver catalase was purchased from Roehringer-Mannheim (specific activity approximately 39,000 units per mg). Argon was ultrahigh purity, ionization grade, purchased from Matheson, and contained less than 1 ppm of O2 and about 0.5 ppm of CO. The argon tank was equipped with a Matheson high purity regulator, model 3500-580, tested to have a leak rate of less than 2 X lo-lo atmospheric cc per s

of helium. All other reagents were the best commercially available.

Methods

Optical Spectra-Optical spectra in the visible and near in- frared were recorded at room temperature on a Cary model 14R recording spectrophotometer using a high intensity source, 0 to 1.0 or 0 to 0.1 optical density slide wires, and l-cm or 2-mm light path silica cells.

Electron Paramagnetic Resonance--EI’R spectra were taken on an X-band instrument which was described by Feher (18), operating between 9200 and 9300 MHz and at pumped liquid helium temperatures (I .5”K). The microwave cavities, which were resonant in the Tl& mode, were constructed of two hobbed brass halves, split along the horizontal current null lines. The interior of the rectangular cavities was electroplated first with gold and then with silver and finally was spray-coated with a thin layer of acrylic resin. The samples contained approxi- mately 100 nmoles of heme in about 0.7 ml.

Atomic Absorption Spectroscopy-This was performed on a Perkin-Elmer model 303 spectrometer operating at 248 rnp or 372 rnp, with Fisher certified ferrous ammonium sulfate and sperm whale myoglobin (see below) as standards.

il!lyoglobin Concentrations-These concentrations were deter- mined by optical spectra in the visible region, by using the extinction coefficients of Hapner et al. (19) for ferric sperm whale myoglobin at pH 6.2, based on dry weights of carefully purified

633 samples: ernM = 3.42, 503 %f = 9.23. These values were quite

insensitive to variations in pH in the pH range 6.0 to 6.5, and were consistent with an tirnN at 540 rnp of about 10.4 for ferric MbCN.

Quantitation of EPR X;oectra-This was performed from a knowledge of the g values and the area under any single feature of the EPR spectrum, using a ferric myoglobin standard at pH 6. The absolute accuracy of this quantitation was .t20% of any single measurement (16).

Total Heme Concentration in Suljmyoglobin samples-Total concentration was determined by three independent techniques: calculation from material balance in the preparation, atomic absorption spectroscopy, and quantitation of the EPR spectrum of ferric sulf-Mb fluoride, which is a single high spin species. The second and third methods gave results agreeing with that of the first to within 3% and 8%, respectively.

Anaerobic Optical Spectra-These were taken using a Thunberg anaerobic cuvette with a side arm bearing a rubber serum stopper and with a double bore stopcock to enable flushing with gas and rapid sealing. With constant gentle shaking, the samples were flushed for 10 to 15 min wit,h argon delivered to the Thunberg stopcock through heavy wall butyl rubber tubing (Fisher) to retard 02 seepage and via an intermediate gas wash- ing bott,le filled with Hz0 to saturate the argon with Hz0 vapor. Apiezon N grease was used on all connections.

Digitalization of Optical Spectra-Digitalization for input to a computer was performed by using an optical line follower which could read the edge of a line to within 0.5 mm (0.002 absorbance unit on these spectra) by sensitivity to black-white contrast. The spectra were recorded in 100 channels of memory (4 rnp per channel) and then punched onto paper tape. Since the line follower could not cross an intersection of lines, such as at grid marks or isosbestic points, individual spectra had to be traced

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Issue of May 25, 1971 J. A. Berxofsky, J. Peisach, and W. E. Blumberg 3369

by hand before use of the line follower. The over-all error in the final digitalized spectra relative to the original data was found to range from less than 1% on maxima and flat, regions to about 3% on the steepest slopes.

Sulfmyoglobin Preparation

Ferrous Xulfmyoglobin-This was prepared by a modification of the methods of Nicholls (10) and of Morel1 et al. (ll), making use of the conditions determined by Wittenberg, Wittenberg, and Kampa2 for obtaining a maximum half-life (158 min at 10’) of MbIV. A typical preparation is as follows.

All procedures were carried out at O-4”, except for observation of optical spectra at room temperature. Sperm whale ferric myoglobin, 0.94 ml of 3.26 mM solution in 0.02 M KPi buffer, pH 7.0, was adjusted to pH 8.0 by the addition of 0.1 ml of 1.0 M

KPi buffer, pH 8.0. To the ferric myoglobin was added a 4-fold molar excess of Hz02 (0.07 ml of a freshly prepared 1:43 dilution of stock 8.7 M I1202 in 0.1 h!I KPi buffer, pH 8.0), producing an immediate change of color from brown to the bright red of MbIV. After 40 set, 1 ~1 (approximately 780 units) of 20 mg per ml of catalase was added to destroy the unreacted HsOs. Thirty-five seconds later, a 1.5.fold molar excess of (NHJ2S (40 ~1 of a fresh 1:37 dilution of 4.3 IM stock scJution in 0.1 M

KPi buffer, pH 8.0) was added, producing an immediate change to the deep bluish green color of ferrous sulf-Mb. As quickly as possible (within 2.3 min) the green solution was placed onto a column, 1 X 36 cm, of Sephadex G-25 previously equilibrated with 0.1 M KPi buffer, pH 8.0. The green material was eluted within 25 min with the same buffer and came off the column in a total volume of 3.7 ml, to give a concentration of 0.80 mM calcu- lated by material balance. Optical spectra were taken im- mediately, since autoxidation of the ferrous protein is noticeable from absorbance ratios after even 1 or 2 hours at 4”. (Prelimi- nary kinetic data give a half-life for the autoxidation at room temperature of about 200 min.) The absorbance ratio Ae17 : AsgO was 2.73, slightly higher than before passage through the Sephadex column. Other preparations, such as those with a large excess of (NH&S as used by Morel1 et al. (II), gave ratios of A617:A580 of no greater than 2.1, while elimination of the catalase and other variations in procedure gave ratios less than 1.0. The use of too little sulfide gave preparations containing an optically observable species recognized as ferric sulf-Mb. NazS can be substituted for (NHJzS in the preparation (see “Discussion”).

Ferric Sulfmyoglobin-This was prepared in the cold by pas- sage of 2.7 mI of the above ferrous sulf-Mb preparation through the same column, 1 X 36 cm, of Sephadex G-25 previously layered with 0.1 ml of a fresh solution of 70 mg per ml of KsFe- (CN)6 (about a IO-fold molar excess) which had been allowed to travel 1 cm down the column before addition of the ferrous sulf- Mb. The protein was eluted at a flow rate of 0.5 ml per min with the same buffer. As the green ferrous sulf-Mb passed through the yellow ferricyanide it turned to the brown color of ferric protein. When the protein reached the bottom of the column, it had left the yellow band of ferricyanide above it, well separated by a white space of 6 cm. The brown ferric sulf-Mb was eluted in 3.95 ml, to give a final concentration of 0.55 mM by

2 J. B. Wittenberg, B. A. Wittenberg, and L. Kampa, personal communication.

material balance. The concentration of iron was confirmed by atomic absorption spectroscopy. The ferric protein was stable by both optical and EPR spectroscopic criteria for days in pH 8 solution at 4”, for 1 or 2 months frozen at, -2O”, and for an indefinite period when quickly frozen and stored in liquid Na (77°K).

RESULTS

EPR Spectra---No EPR could be ascribed to ferrous sulf-Mb. At pH 6.0, ferric sulf-Mb has both high spin and low spin heme when studied at 1.5”K. The high spin species (Fig. 1) with an EPR absorption extending from g = 6 to g = 2 (20, 21) is es- sentially axial in that no splitting of the g = 6 resonance can be observed in frozen solution. A low spin form, not identical with the hydroxide (see below) or any other form that we have studied, has been observed with g values of 2.50, 2.26, and 1.83 (Fig. 2). Preliminary optical and EPR evidence suggests that the popula- tion of this low spin species is temperature-dependent, and that

I MAGNETIC FIELD

FIG. 1. EPR absorption derivative spectra of high spin forms of ferric sulfmyoglobin. The upper trace in each case is a 5000 gauss sweep. Below each of these is a 1000 gauss sweep of the 9 = 6 region to show relative broadening. The arrow indicates g = 2.0. The spectrometer gain is different in all six spectra. k, ferric sulf-\/lb, 0.25 mM, buffered at pH 6.2 in 0.5 M K$i. B, ferric sulf-Mb fluoride. nreuared bv the addition of a solution of 70 mg of KF.2HzO dissoiveb in 0.5”ml of 0.1 M KPi, pH 6.0, to 0.2 ml of 0.48 mM ferric sulf-Mb buffered at pH 8.0 with 0.1 RI KPi, immediat,ely before freezing. The g = 2 end of the high spin signal is split into a doublet by the F- nucleus, but, since this signal is riding on that of cubic iron at 9 = 2.04, the spectral splitting is hard to observe at this gain, but is easily recognized at higher gain and expanded sweep. C, ferric sulf-Mb azide, prepared by addition of 0.2 ml of 0.55 mw ferric sulf-Mb to a freshly prepared solution of 10.2 mg of NaN3 in 0.45 ml of 0.2 hx

KPi, pH 6.0, immediately before freezing.

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3370 Xdfheme Proteins I: Xu(fmyoglohirk Vol. 246, No. 10

1.65

>

2.61 2.23

/&;;,:,I D

MAGNETIC FIELD

FIG. 2. EPR absorption derivative spectra of low spin forms of ferric sulfmyoglobin. Spectra were taken at 1.5”K at a sweep width of 2500 gauss. The spectrometer gain was different for each spectrum. The spectra are aligned at g = 2.0, usually marked by the high field end of the spectrum of residual high spin material. The resonance around g = 2.04 is ascribed to a small amount of cubic Fe3+ contaminant. A, low spin component of ferric sulf-Mb at pH 6.2, prepared by the addition of 0.35 ml of 1.0 M KPi, pH 5.9, to 0.3 ml of ferric sulf-Mb, 0.55 mM, in 0.1 M KPi, pH 8.0. B, ferric sulf-Mb hydroxide, prepared by the addi- tion of 0.5 ml of 1.0 M KPi, pH 10.2, to 0.2 ml of 0.48 mM ferric sulf-Mb in the cavity immediately before freezing. The minor component indicated by ~TTO’WS at g = 2.61, 2.16, and 1.82 is as- cribable to ferric Mb hydroxide, representing about 10% of the total heme. C, ferric sulf-Mb cyanide, prepared by the addition of 1 mg of solid KCN to 0.7 ml of 0.14 M ferric sulf-Mb buffered at pH 6.6 with 0.1 M KPi, immediately before freezing. D, ferric sulf-Mb azide, prepared by the addition of a solution of 10.2 mg of NaN3 in 0.45 ml of 0.2 M KPi, pH 6.0, to 0.2 ml of ferric sulf-Mb, 0.55 rnM.

at room temperature the pH 6.0 ferric sulf-Mb is essentially all high spin. This temperature-sensitive spin state conversion of

this protein will be the topic of a future communication. As the pH is raised, the EPR signal near g = 6 diminishes and a new

resonance (g = 2.44, 2.20, 1.88), attributable to ferric sulf-Mb hydroxide, appears. With the diminution of the g = 6 reso-

nance, the EPR absorption derivatives at g = 2.50, 2.26, and

1.83 also decrease in intensity.

The addition of fluoride to ferric sulf-Mb yields a single high spin compound having an EPR absorption also extending from

g = 6 to g = 2 (Fig. 1). However, it will be noted that the g = 2 end of the high spin spectrum shows a splitting of about

48 gauss due to the nuclear spin of the bound I? and diagnostic

for fluoride binding to heme iron (21). Similarly, the g = 6

signal of the fluoride, with a width of 59 gauss between derivative extrema, is broadened relative to the ferric sulf-Mb at pH 6,

TABLE I

EPR g values of low spin compounds of ferric suljmyoglobin and

Ligand

OH- (pH 10-11). x3-.... CN-... :: .:.. SWc

I-

ferric myoglobin

Ferric sulfmyoglobin Ferric myoglobin

61 92 93 gi 52 93

2.44 2.20 1.88 2.60 2.15 1.83 2.61 2.23 1.80 2.82 2.19 1.71a 2.65 2.43 1.65 3.5 1.9 <0.756 2.38 2.26 1.91 2.58 2.25 1.83

a Data from HelckB, Ingram, and Slade (22). b J. Peisach and W. E. Ulumberg, unpublished observation. c The assignment of SH- as the ligand is questionable, since

these samples, prepared by the addition of (NHb)$ to the protein solutions in the EPR cavities immediately before freezing in liquid Nz, contained several low spin species and also EPR silent ma- terial, presumably reduced protein. The g values of the majority species are gix en in each case.

1 , , .I , , / , I I I ( 1 ( ( ( IL_.L_i

440 480 520 560 600 640 680 720 760

/

i

WAVELENGTH, mp

FIG. 3. Acid-base titration of ferric sulfmyoglobin in the visible region. The sample was 2.6 ml of a 0.085 mM solution of freshly prepared ferric sulf-Mb in 0.1 M KPi buffer. Changes in pH were effected by addition of microliter volumes of NaOH. At pH 6.32, ern~ = 3.3 at 717 rnp. At pH 10.9, ern~ = 7.7 at 672 rnp.

which has a width of only 39 gauss. The g values of several low

spin derivatives of ferric sulf-Mb and ferric Mb are summarized in Table I.

pH Titrations-Another measure of the electronic environ-

ment of the heme is the pK of the equilibrium

Heme Fe3+ Hz0 ti heme Fe3+ OH- + IIf

To determine this pK for ferric sulf-Mb and ferric Mb, acid-base titrations were performed in the visible and the near infrared

regions. The spectral changes of ferric sulf-Mb in the visible show clean isosbestic points within the pH range 6.32 to 10.0 (Fig. 3). Above pH 10, deviations from isosbestics appeared,

indicative of the formation of another species. The plots of optical density against pH in Fig. 4 show that identical pK

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7.0 8.0 PH

9.0 10.0 11.0

FIG. 4. Analysis of visible spectral acid-base titration of ferric sulfmyoglobin, based on the spectra of Fig. 3. A, optical density at 672 rnp, an absorption maximum of the alkaline form, versus pH. B, optical density at 718 rnp, an absorption maximum of the acid form, versus pH. Both forms are seen to vary with an identical pK of 8.45 f 0.05.

values are obtained from measurement at 672 and 718 rnp, ab- sorption maxima of the alkaline and acid forms, respectively. This result indicates that the interconversion of only two species is being observed in the titration. The near infrared spectra of the high and low pH forms of both ferric sulf-Mb and ferric Mb are shown in Fig. 5 (cf. ferric Hb (23)). For ferric s&Mb, both the visible and the near infrared titrations gave a pK of 8.45 f 0.05. This value was confirmed by a computer analysis of the visible titration data described below, which gave a value of 8.44 to 8.48. Both the near infrared and the visible titrations of ferric Mb gave a pK of 8.9 to 9.0. which confirms the values of 8.9 (24) and 9.0 (25) previously published. Clearly, then, there is a difference in the pK of ferric sulf-Mb from that of ferric Mb of about 0.5 pH unit, equivalent to a factor of greater than 3 in the equihbrium constant for an optically observable proton dis- sociation, presumably from water bound at the heme.

Determination of Purity: Nature of Minority species-All preparations of ferrous sulf-Mb, characterized optically by an intense absorption maximum at 617 to 618 rnp, contain varying amounts of a contaminant with maxima at 545 and 580 rnp (10, 11). Several lines of evidence serve to identify this contami- nant. As can be seen from Fig. 2, the EPR spectrum of a sam- ple of ferric sulf-Mb taken to high pH shows the spectrum of a minority species having g values clearly different from those of ferric sulf-MbOH, the majority species. The g values of this minority species (2.61, 2.16, 1.84) are the same as those for ferric Mb hydroxide (26) (cf. Table I). The presence of the contami- nant is once more observed as a minority component (g = 2.82,

WAVELENGTH, m,w

FIG. 5. Near infrared spectra of ferric myoglobin and ferric sulfmyoglobin preparations. A, ferric &f-Mb, 0.45 mM, in phos- phate buffer. Dashed curve, pH 6.8, ern~ = 0.49 at 1050 rnp. Solid curve, pH 10.5, obtained by addition of microliter volumes of NaOH to thelower pH material. CAM = 0.55 at 975 rnp; crnn = 0.71 at 825 mM. The values given here for ELM are uncorrected for the presence of 10% ferric Mb (see text). B, ferric Mb, 0.47 mM, in phosphate buffer. Dashed curve, pH 7.35. ELM = 0.83 at 1030 rnp. Solid curve, pH 10.1, obtained by addition of microliter volumes of NaOH to the lower pH material. ernn = 0.68 at 825 mr; E~X = 0.50 at 740 mw.

2.25, and 1.69) in the azide preparation of ferric sulf-Mb. These g values are similar to those of ferric Mb azide. Thus we may conclude that the contaminant in the ferric sulf-Mb is ferric Mb. This assignment is consistent with our observations of the 580 and 545 rnp absorbing material in ferrous sulf-Mb preparations (see below). The positions of these maxima are very near those for MbOz (543, 581 mp). We believe Mb02 to be the major contaminant in the ferrous sulf-Mb preparation, and it is from this compound that both ferric Mb hydroxide and ferric Mb azide are derived which appear as minority species in the EPR after ferricyanide oxidation. This oxidation of the ferrous sulf- Mb preparation completely eliminates the maxima at 545 and 580 rnp, as would be expected if they were derived from MbOz. Flushing the ferrous sulf-Mb preparation with oxygen-free argon for 10 min, or anaerobic addition of sodium dithionite, shifts the 580 rnp maximum to about 570 rnp. This observation is con- sistent with a partial conversion of the MbOz contaminant to the deoxy form with a maximum at about 560 mp. Addition of (NH&S to ferric Mb produces a species with A,,, = 542, 579 mp. However, the above observations are not consistent with the hypothesis that the contaminant is ferric Mb sulfide.

EPR Quantitation-The well resolved EPR signals of ferric sulf-MbOH and ferric MbOII provide an excellent measure of

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3372 Xulfheme Proteins I: Sulfmyoglobin Vol. 246, No. 10

0.0 ’ ’ ’ ’ ’ 1 ’ ’ ’ ’ 1 ’ ’ ’ ’ ’ ’ ’ ’ ’ 1 ’ 1 1 1 I 500 550 600 650 700 7:

WAVELENGTH, mu

FIG. 6. Computed spectra of pure ferric sulfmyoglobin. A, alkaline form, pure, computed as described under “Results.” B, comparison of computed best fit curve (solid) to the observed spec- trum (dashed) at pH 8.51, near the pK. C, acid form, pure, com- puted as described under “Results.” All curves except the lower- most (C) are arbitrarily displaced on the ordinate. The base line for each spectrum is 0.4 unit of crn~ (one scale division) below the lowest point on the curve.

the relative concentrations of the two compounds in the ferri- cyanide-oxidized sulf-Mb preparation when brought to high pH. Quantitation of the EPR spectra, recorded as the first derivative of absorbance, can be done by double integration. Although the absolute accuracy of this quantitation is only about &20%, the relative amounts of two species in the same sample can be determined to within about 10%. Measurements by this method of the two species in our alkaline ferric sulf-Mb samples consistently indicated that 10 to 12% of the tota,l low spin ma- terial at pH 10 to 11 was ferric MbOH. Variations in the pre- parative procedure for sulf-Mb, such as those used by Morel1 et al. (II), yielded a greater percentage of ferric MbOH.

Computer Analysis of Optical Titration3-Owze the identity of the contaminant was known, its optical spectra and those of the sulf-Mb preparation at various values of pH, obtained from the titrations described above, could be used in a least squares fitting program to find the fractiona purity and the pK of the ferric sulf-Mb. Simultaneous equations of the form

etotal = F[fpHEA + (1 - fpH)d + (1 - &Mb

were used, representing the observed molar extinction etotal of the mixture at a given pH and wave length X as a function of the

3 Complete listings of both programs will be given in the dis- sertation to be submitted by J. A. B. to the Albert Einstein Col- lege of Medicine of Yeshiva University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

24.0

1

20.0 -

0.0 ’ ’ , , , ,

500 550 600

WAVELENGTH, mp

FIG. 7. Visible spectrum of ferrous sulfmyoglobin. Dashed curve is the observed spectrum of an actual fresh preparation of ferrous sulf-Mb, shown to contain about 90% ferrous sulf-Mb and 10% Mb02 (see text). Solid curve is the computed spectrum of pure ferrous sulf-Mb generated from the dashed curve assuming the above composition and the known spectrum of MbOz.

fraction F of the preparation which is ferric sulf-Mb, the fraction

f PH of the ferric sulf-Mb in the acid form at the given pH, the extinctions CA and es of the acid and alkaline forms of ferric sulf-Mb at the given wave length X, and the observed molar extinctioneMb of ferric Mb measured at that pH and wave length.

No assumptions about the pK of either ferric sulf-Mb or ferric Mb were needed, except that the spectra around pH 10 to 10.9 and below p1-I 6.3 were assumed to be those of the pure alkaline and acid forms, respectively. A refinement involving successive approximations was used to correct for error in the observed high pH spectra due to denaturation.

The fitting operation was applied at three intermediate values of pH, 7.96, 8.51, and 9.12. The computed best fit values of

f pH at these three values of pH were 0.754, 0.460, and 0.187, re- spectively. These were equivalent to pK values of 8.45, 8.44, and 8.48, so that the value of 8.45 f 0.05 determined experi- mentally (Fig. 4) was confirmed. The computed spectra of the pure acid and alkaline forms of ferric sulf-Mb based on this pK are shown in Fig. 6. These were obtained by simultaneous solution of the functions defining the optical spectra of ferric Mb and ferric sulf-Mb at two intermediate values of pH. Also shown for comparison are the computed and observed spectra at pH 8.51. The goodness of fit was even better at pH 7.96 and pH 9.12 than at pH 8.51, which is near the PK.

The computed best fit values of fractional purity, F, were 0.94, 0.95, and 0.98 for the above three values of pH, giving a mean of 0.96, or 96% purity. This result is to be compared with 88 to 90% from the EPR quantitation. Although the mean of these values is 92%, since the EPR measurement is more direct we would tend to give it more weight, and to accept a minimum value of 90% sulf-Mb in our preparation.

We did not attempt further purification of this preparation, since preliminary attempts at electrofocusing and polyacrylamide gel clcctrophorcsis did not result in good separations.

Optical Spectrum of Petrous Xulfmyoglobin-The spectrum of pure ferrous sulf-Mb was important to compute, since this was

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0.2 - t $ o.:-

z oo,o I I II./ ! i I / I I I I I -

co/ 460 500 540 580 620 660 700 740 780

WAVELENGTH, m,i

FIG. 8. Optical spectra of low spin derivatives of ferric sulf- myoglobin. A, azide, prepared by addition of 10 ,LJ of a freshly prepared solution of 0.4 mg per ml of NaNa, t,o 1.0 ml of 0.083 miv ferric sulf-Mb buffered at pH 6 with 0.1 M KPi. ~,a: 706 rnp, 2.2; 623 rnp, 10.2; 593 rnp, 9.4. B, cyanide, prepared by addition of less than 1 mg of solid KCN to 1.0 ml of a 0.083 mM solution of ferric sulf-Mb, buffered at pH 6 with 0.1 M KPi. ELM = 11.2 at X,,= 594 rnp. The values given here for crn~ are uncorrected for the presence of 10% ferric Mb.

the form used to assay concentration and purity of sulf-Mb by all previous workers. In accordance with the demonstration above that our preparation was 90% pure and that Mb02 was the impurity, the spectrum of pure ferrous sulf-Mb was ob- tained by subtraction of a spectrum of pure MbOz, scaled to cor- respond to 10% of the total heme, from the observed spectrum of the ferrous sulf-Mb preparation, followed by renormalization. The resulting spectrum, with a millimolar extinction coefficient at 617 rnp of 24.0, is shown in Fig. 7, along with the original uncorrected observed spectrum of our preparation.

Optical Spectra of Other Compounds of Sulfmyoglobin-The optical spectra of ferric sulf-Mb cyanide and azide are shown in Fig. S. In contrast to the findings of Nicholls (lo), we have found that the optical spectra of these derivatives in the visible region are stable for over 1 week at room temperature. As ex- pected, cyanide and azide, in concentrations sufficient to saturate the ferric protein completely, showed no effect on the spectrum of the ferrous protein. Moreover, the rate of autoxidation at 4” of the ferrous protein was not significantly affected by the presence of these ions. Table II gives the positions of the Soret maxima and approximate extinction coefficients for ferric sulf-

TABLE II

comparison oj Soret maxima of compounds of ferric and jerrous sulfmyoglobin and myoglobin

Compound

Sulf-Mb

&3x %Ma

ma

High spin (acid) ferric.. 404 117 Ferric hydroxide, 408 100 Ferric azide. 412 85 Ferric cyanide.. 412 73.7c Ferrous . . . . 421 86

- Mb

LIlaa frnM

WJ

409.5b 1577J 4146 97.2b 423 110 422 109.7* 434d 105d

a Uncorrected for the 90yo purity of the preparation. b Data of Hanania, Yeghiayan, and Cameron (27). c Shoulder at 412 m@. See text. d Data of Keilin and Hartree (28).

Mb and Mb bearing various ligands, as well as for the ferrous forms. All of the Soret maxima of the derivatives of sulf-Mb have only slightly greater peak widths than their Mb counter- parts. The greater widths are due to the presence of shoulders ascribable to the corresponding derivatives of the 10% ferric Mb contaminant. However, in the case of the ferric sulf-Mb cyanide preparation, the absorbance at 422 rnp, the position of the Soret band of ferric MbCN, was actually 8% larger than that of the shoulder at 412 rnp, which we ascribe to ferric sulf- MbCN. Since this last compound is considered to be 90% of the preparation, and since it does not appear to be converted significantly to ferric MbCN by the cyanide as judged by the spectrum in the visible region, we must assume that the extinc- tion coefficient of ferric sulf-MbCN is considerably lower than that of ferric MbCN in the Soret region.

DISCUSSION

The computed spectrum of ferrous sulf-Mb (Fig. 7) shows a new absorption maximum at about 570 rnp, much less intense than the most prominent absorption in the visible at 617 ml*. These maxima are separated by an energy of 1360 cm-l, which is almost the same as the 1500 cm-l separation of the shoulder of deoxy-Mb around 515 rnp from the main absorption at 558 mp. This new observation is consistent with the characteriza- tion of ferrous sulf-Mb as the deoxy rather than the oxy protein, since flushing with oxygen-free argon for 15 min or reduction with dithionite does not noticeably change the 618 rnb absorp- tion, and has only the effect of unmasking the 570 rnp absorp- tion upon reducing the Mb02 impurity, which absorbs at 580 rnp, to deoxy-Mb, which absorbs at 558 mp.

The visible spectrum of ferric sulf-Mb azide suggests the presence of some residual high spin material absorbing at 717 and 595 mp. Even at 1.5”K, and in the presence of a large excess of N3, there remains high spin material as judged by the EPR spectrum of Fig. 1. However, the g = 6 resonance in the azide preparation is significantly broadened, having a width of 73 gauss between derivative extrema as compared with 39 gauss for the g = 6 resonance of ferric sulf-Mb at ~1% 6. This broaden- ing suggests that the high spin material in the azide preparation may be a high spin azide derivative rather than residual starting material with H20 as ligand. Broadening due to ligand exchange is ruled out since there can be no exchange at 1.5”K.

Our computed spectrum of ferrous sulf-Mb (Fig. 7) also allows

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3374 Sulfheme Proteins I: Xulfmyoglobin Vol. 246, No. 10

TABLE III

Comparison of purity of different preparations oj sulfwlyoglobin and sulfhemoglobin

Source %lM, 617 mfi

--

This study-computed pure ferrous &f-Mb (Fig. 7) 24.0

This study-preparation de- termined to be 90% pure 21.6

Morel1 et al. (11). 17.8 Nicholls (10) 17.5 Drabkin and Austin (7)a. 10.6 Michel (2). 18.3b

$&/M/24.0 Ratio: Ratio/ A6l,:A581 3.28

-

1.00 3.28 1.00

0.90 2.96 0.90 0.74 2.38 0.73 0.73 1.87 0.57 0.44 1.33 0.38 0.76 1.66 0.51

a These data arc for sulf-Hb, which is included in this table on the assumption that the values are comparable to those of sulf- Mb. This assumpt,ion is justified by the recent preparation of sulf-Hb by Nichol et al. (4) withAelT:Acs,, = 2.41.

b Calculated from the quoted specific absorption coefficient, E,,v = 1.03, in Reference 2 assuming a molecular weight of 17,800 (29).

an estimate of the purity of earlier preparations. An early criterion for purity was that of Morel1 et al. (ll), who used the absorbance ratio A618:A580. By this criterion, their preparation, with a ratio of 2.12, was more than that of Nicholls (lo), with a ratio of 1.86, but was less pure than the one reported here, with a ratio of 2.73. However, since 580 rnp is not an isosbestic be- tween Mb02 and ferrous sulf-Mb, this ratio is not a linear func- tion of purity, or of per cent sulf-Mb in the preparation. In the spectra of Fig. 7, and others not shown, representing mixtures of varying proportions of MbOZ and ferrous s&-Mb, there are several isosbestic points, the most convenient being at 561 mp. Hence, since the ratio Ao17:A5e1 for pure Mb02 is nearly zero (~0.026), the ratio As17 :Asel should be approximately propor- tional to per cent purity. This criterion can be applied to all samples in which the contaminant is MbOz, namely those pre- pared by methods similar to the present one. A second criterion of purity is the computed millimolar extinction coefficient at 617 rnp for ferrous sulf-Mb. These extinction coefficients, measured from published spectra, and their ratios to the revised value of 24.0 are given in Table III, along with the ratios Ac17: A 561.

In the light of this optical spectral analysis, some earlier re- sults of other investigations may be re-evaluated. Most authors of the late 1930’s and 1940’s used the extinction coefficient of Drabkin and Austin (7) for ferrous sulf-Hb, which is now seen to be low by a factor of 0.44, assuming that the molar extinctions for sulf-Mb and sulf-Hb are comparable (see Footnote a to Table III). Correction by a factor of l/O.44 resolves the dis- crepancy which Nijveld (30) found in the stoichiometry of Michel’s reaction mechanism (S), and also the factor of 2 dis- crepancy in ferricyanide consumption in Nijveld’s own oxidation- reduction titration of sulf-Hb (30).

The result of Morel1 et al. (ll), who concluded that 1.5 g atoms per hcme of 35S were incorporated in sulf-Mb, should similarly be revised by a factor of l/0.73, based on the purity of their pro- tein, to give a total incorporation of 2 atoms of sulfur per heme. Of these, as much as 0.7 g atom per mole was found bound to the globin, while the radioactivity (59%) that was cleaved from the protein by acid-acetone did not appear to be a single species, a large fraction separa,ting from the heme on extraction. The

revised figures render these results inconclusive in determining the site of bound sulfur.

We have prepared ferrous sulf-Mb substituting 1.6 moles of NazS per mole of heme in place of (NH,)&. Since Na&‘. 9Hz0 is a weighable, albeit hygroscopic, solid, the upper limit of con- centration of sulfide can be known with greater certainty than with (NHSZS. Use of less sulfide than about 1.5 moles per mole of heme leads to the formation of a significant amount of ferric sulf-Mb along with the ferrous material. These observa- tions are consistent with Nicholls’ results (10) that ferric sulf-Mb is first formed with the addition of 1 mole of sulfide per heme, and that addition of excess sulfide subsequently reduces the ferric protein to ferrous. We conclude, furthermore, with this stoichiometry of 1.5:1 that (a) 0.5 mole of sulfide provides at least 1 reducing eq in the reduction of ferric sulf-Mb to ferrous, since only 0.5 mole of sulfide in excess is necessary for complete reduction, and therefore (b) not more than 1 g atom of sulfur is available to be specifically bound per mole of Mb converted to s&-Mb. This last conclusion is to be compared to the %in- corporation study of Morel1 et al. (II), who found more than 1

sulfur atom to be bound when they used over a loo-fold excess of labeled sulfide in their preparation.

The physical studies presented here throw some light on the question of the location of the sulfur atom in sulf-Mb. The location of sulfur has been suggested by Lemberg and Legge (3) to be on the proximal histidine ligand of the heme in the form of thiohistidine. Morel1 et al. (II), on the other hand, have suggested on the basis of their experiments that the sulfur atom resides on the porphyrin of the molecule, as a chlorin episulfide. A third possibility, suggested by Nicholls (lo), is that a thiol substitutes for a proton attached to a methene carbon. A final possibility, also considered by Morell,4 and for which we produce evidence on the basis of these physical studies, is that 1 molecule of HzS adds across a p-0 double bond of a porphyrin pyrrole, leading to the formation of a thiochlorin.

As has been pointed out by Morel1 et al. (ll), the optical spectra of some sulf-Mb derivatives resemble those of metal- lochlorins. A metalloprotoporphyrin IX, such as heme, with almost electronic equivalence in the z and y directions in the porphyrin plane and thus Ddh symmetry, exhibits a Soret, ab- sorption near 400 rnp and two absorption peaks in the visible. In contrast, in a metallochlorin, the two visible absorption peaks are split to four (31), since the symmetry is lower than D4h. Of these, the maximum at the longest wave length is the most in- tense in divalent metal compounds. The optical absorption maxima from our studies which can be ascribed to a chlorin structure are summarized in Table IV.

The difference in energy between the Soret maximum (denoted B) and the cy peak, the longest wave length absorption in the visible region (the vibrationless &(0-O) band in Platt’s notation (32)), is a measure of the configuration interaction (34) between the first two optically excited states of the porphyrin a-elect,ron system which have an accidental degeneracy. As can be seen from the data of Smith and Williams (35) (Table V), for ferric myoglobin derivatives, there is a continuous decrease in the configuration interaction energy with increase in electron- withdrawing capacity of the exchangeable sixth ligand of the heme. As shown by Hill and Morallee (37), electron-nithdrau- ing z ligands of heme lower the r-electron density of the por-

4 D. B. Morell, personal communication.

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phyrin and it is in these compounds that there is the smallest difference in energy between Soret maxima and o! peak and the least configuration interaction.

A similar analysis can be performed for metallochlorin spectra by using the average of the vibrationless absorptions in the visible region (i.e., Q&O) and &,(0-O)) in place of Q(O-0) (Table V). In both the metal-containing (36) and the metal-free tetraphenylporphin (36) systems, the conversion of the porphin to a chlorin raises this ener,y difference B - Q by the same quan- tity, 420 cm+. The data in Table V show a similar increase from the Mb to the sulf-Mb compounds varying from 330 cm+ to 540 cm-’ and is thus consistent with a porphyrin to chlorin transformation. On the other hand, in order for the increase of configuration interaction of sulf-Mb compounds to be brought about by changing the composition of the fifth ligand of the porphyrin, the ligand would necessarily have to be more electron- supplying than histidine. Thus the thiohistidine structure postu- lated by Lemberg and Legge (3) would be in contradiction to the known behavior of thiols. The direction of the electron- withdrawing effect of a sulfhydryl group bound to an aromatic system (porphyrin or histidine) can be inferred from its effect on pyridine. According to Perrin (38), a 4-mercapto group lowers the pK of pyridine from 5.25 to 1.43, in the same direction as the known electron-withdrawing 4-cyano group, which lowers the pK to 1.90. In contrast, the known electron-donating 4- amino group raises the pK to 9.25. Therefore, a sulfhydryl group in this location is electron-withdrawing, in agreement with the more general discussion by Pryor (39), and we conclude that the sulfur atom of sulf-Mb cannot be bound to the proximal histidine ligand of the heme. This contention is further sup- ported by attempts to insert a sulfur atom on to histidine bound to heme by using space-filling Corey-Pxuling-Koltun models. The atomic volume of sulfide is so large that binding to the proximal histidine carbon atom 01 to the N--Fe bond cannot be accomplished without a great deal of strain.

EPR spectroscopy of ferric heme proteins is a direct probe of the electronic environment of the heme iron, i.e. its ligand field. For the low spin forms shown in Table I, the ligand field can be analyzed by the approach of Blumberg and Peisach (16). The three y values determine two crystal field parameters, which are chosen to be one of tetragonal and one of orthorhombic sym- metry, as dimensionless ratios, 1 A/x j and 1 V/X 1, to the spin orbit coupling energy X. The tetragonality, 1 A/x 1, is a meas- ure of the electron donation of the z-y ligands (porphyrin pyr- role nitrogen atoms) relative to that of the two z ligands. The ratio of j V/A 1, the rhombicity, is a function of geometry alone. As shown by Blumberg and Peisach (16), if one plots / V/A 1 against j A/h 1 for a large number of protoheme protein deriva- tives, one finds that correspondin g derivatives of different pro- teins group together in regions shown in Fig. 9, in which the positions of various s&-Mb derivatives and the corresponding Mb derivatives are indicated. It can be seen that the sulf-Mb derivatives fall outside the envelopes describing the analogous compounds of Hb, Mb, and cytochromes (16)) consistently below and to the right of the corresponding Mb derivatives. The cyanide derivatives do not fall in any of these envelopes, and, in fact, the EPR spectra of CN- derivatives of ferric heme pro- teins are generally very broadened and therefore hard to study. However, ferric HbCN, MbCN, and cytochrome c CN have similar crystal field parameters and therefore similar structures. In contrast, as shown in Fig. 2, the CS- derivative of ferric

TABLE IV

Assignment of optical absorptions of sulfmyoglobin a,nd its derivatives

The notation used follows Platt (32), and the absorption maxima are given in wave numbers. The Sorct absorption is denoted by B and the visible absorptions are denoted by Qz and Qy. The O-O and O-l designations refer to electronic excitations involving zero and one simrdtaneous vibrational excitation, re- spectively.

I QZ

Ferrolls sldf-&lb. _. Ferric sulf-Mb, high

spin. Ferric sulf-.&bOH. : Ferric sulf-MbNI.. Ferric sulf-MbCN.

QU B

o-o o-1 o-o O-l ~-~

16,210 17,540 18,35C~ 19,cino 23,750

13,930” 16,860b 24,750

16,020 17,422c 18,020 19,23Cc24,510

16,080 lG,86od 18,180 19,310 24,270 16,810 18,18Uc 18,870 I 24,270

u Assumed to be underlying the absorption of the Mb02 con- taminant.

b Assignment uncertain. Possibly a charge transfer transit,ion as in ferric Mb (33).

c Observed in spectrum taken at 80°K. d Assumed to be underlying the absorption of the high spin form

of &f-Mb present.

TAHLE V

Relation between optical transilions in visible and those in new ultraviolel regions for derivatives of myoglobin, sulfmyoglobtin,

and tetraphenylporphin

The notation used follows Platt (32) and the absorption maxima are given in wave numbers. The Q transitions for the compounds not having D4h symmetry are the average of the Q, and Q, vibra- tionless (O-O) transitions.

MbCw MbNIa MbOIIa. MbNOz? MbSCNa.. TPP-free base”. TPC-free basec. Ferric TPPb.. Ferric TPCd

C‘ompound B B-Q

23,700 17,160 6,540

23,800 17,200 G,GOO

24,400 17,240 7,160

24,400 17,060 7,340

24,450 16,860 7,590

23,810 17,200 6,610

23,810 16,780 7,030

23,530 17,390 6,140 24,270 17,710 6,560

Sulf-MbNa .......... Sulf-MbOH .........

24,270 17,130 7,140 24,510 17,020 7,490

a After Smith and Williams (35). 6 TPP (tetraphenylporphin) and TPC (tetraphenylchlorin)

after Dorough, Miller, and Huennekens (36). c After Dorough and Huennekens (31). d A. Adler, private communication.

sulf-Mb has very sharp resonances with well defined g values (Table I), which correspond to a position again far below- and to the right of the other cyanides.

The differences between the crystal field parameters for the low spin compounds of ferric sulf-Mb and the analogous com- pounds of Mb can best be interpreted by noting their effect on the d electron orbitals (see Scheme I). In the case of ferric Mb

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Xulfheme Proteins I: Xulfmyoglobin Vol. 246, No. 10

HbCN

-MINORITY

00’ I I I I I 2 3 4 5 6 7

TETRAGONAL FIELD, IA/h 1

FIG. 9. EPR crystal field analysis, showing the crystal field environment of the heme iron of various ferric Mb and ferric sulf- Mb derivatives (16). The parameters j V/A 1 or rhombicity and 1 A/x 1 or tetragonality, described in the text, are computed from the g values given in Table I and in the text. The boundaries shown are empirically determined envelopes delimiting the areas in which certain types of derivatives group. The region labeled H contains most compounds with an aromatic or imino nitrogen sixth ligand and a histidine fifth ligand, e.g. azide derivatives or dihisti- dine hemichromes (40). The region labeled 0 contains mostly compounds with oxygen sixth ligands, su’ch as hydroxides. The region labeled P, named after cytochrome P-450, contains that compound and other heme mercaptides. The C region is named after cytochrome c, which has z ligands methionine and histidine, and the B region is named after cytochromes bs and bg, although the distal ligand in these cytochromes is unknown. Such a dia- gram can be used to predict from the EPR the nature of the ligands in a low spin ferric heme protein derivative whose structure is otherwise unknown.

6.73x- YZ 6.37x- YZ

,ANTIBONDING ------ _-----

3.84x - xz 3,43x--x2 T

5.1X

NONBONDING O- XY O- XY 1

MbN3 SMbN3

SCHEME I. cl-orbital diagram for azide derivatives of ferric sulfmyoglobin and myoglobin.

azide, which has been studied in a ferric myoglobin single crystal (22), the zz and yx orbitals which are rr-antibonding are split by 2.5 X. This energy splitting has been attributed (41) to the inequivalence of the rr orbitals of the nitrogen atom of the prox- imal histidine bound to the heme iron. The average of these a-antibonding orbitals lies 5.1 X above the nonbonding zy orbi- tal. I f we make the same geometrical assignments of the orbitals for ferric sulf-Mb azide, an assumption which can be verified with single crystal studies, then we find that the average of the n-antibonding orbitals is also just 5.1 X above the nonbonding orbital, while the splitting between them is increased to 3.3 X in accord with the presence of another mode of inequivalence in the z and y directions, namely that of the a-electron system of a chlorin, where the direction which includes the saturated pyrrole is inequivalent to the direction perpendicular to it. Moreover,

the EPR results are inconsistent with the thiohistidine structure as this structure would result in decreased splitting between the nonbonding and the a-antibonding orbitals. Similar arguments hold for the hydroxide derivatives of ferric sulf-Mb and Mb. We are then left with the alternative of placing the sulfur atom at the periphery of the porphyrin.

Since the structure involving substitution on a methene carbon does not involve a disruption of the ring conjugation, its optical spectrum would show little or no departure from the Dlh sym- metry of heme. Moreover, attempts to build this structure with Corey-Pauling-Koltun space-filling models demonstrate that sulfur addition to a methene carbon atom is impossible without disruption of the structure.

Thus we are led to believe that sulfur is bound to a P-carbon of a pyrrole. One consequence of the addition of an electron- withdrawing group at this location would be decreased electron density at each of the z ligands (42). In high spin ferric sulf-Mb each of these z ligand atoms (oxygen from water and nitrogen from histidine) would contain less electron density than in high spin ferric Mb. The pK of the oxygen atom would thus be decreased and this prediction is consistent with the observed pK of ferric sulf-Mb, which is 0.5 pH unit lower than that of ferric Mb. The effect on the proximal imidazole ligand can be studied with high resolution proton magnetic resonance (43) in an anal- ogous manner to the electron density redistribution studies of Hill and Morallee (37).

Without model studies, EPR cannot differentiate between a thiochlorin structure and the chlorin episulfide structure of Morel1 et al. As shown in Table IV, not only the ferrous form, but also all of the ferric derivatives of sulf-Mb have the optical spectrum of a species having less than Dllh symmetry, and in fact the Q, and Q2/ splittings lie in the same range (2000 to 3500 cm+) as those of tetraphenylchlorin free base (2800 cm-‘) (31) and ferric tetraphenylchlorin (3420 c111+).~ If sulfur were added to the porphyrin as an episulfide, in analogy to the re- placement of a double bond of a conjugated polyene by an epox- ide or cyclopropyl group, the ring conjugation of the pyrrole would not be broken, and the optical spectrum would show little or no departure from the Dqh symmetry of the heme.

The thiochlorin structure discussed here is also consistent with the finding of Morel1 et al. (11) that acid-acetone treatment of their preparation of sulf-Mb led to the recovery of ferric proto- porphyrin IX. As is well known, small molecules, such as HOH, added across a double bond of a conjugated polyene, are readily eliminated in acid in order to restore the conjugation. Separa- tion of chlorin from sulf-Mb under less harsh conditions would be required to establish the chemical nature of this heme deriva- tive.

Although X-band EPR cannot be used to differentiate high spin (X = 2) and low spin (S = 0) ferrous heme, it is believed that ferrous sulf-Mb is high spin. Since the electron density has been shifted away from the iron, the cubic crystal field split- ting will be smaller than the splitting in ferrous Mb, which is itself too small to promote the pairing of the electrons into the low spin state. This problem could be resolved by studying the magnetic susceptibility of this material.

Acknowledgments-The authors wish to thank Mr. David Zuckerman for advice and assistance in the electronic and cryo- genic problems in EPR spectroscopy and in the use of the optical

6 A. Adler and P. Hines, personal communication.

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line follower which he designed. The authors also thank Miss 19. Rhoda Oltzik for her preparations of sperm whale ferric myo- globin, Dr. Gene A. Morrill for his advice and assistance in carry-

20,

ing out the atomic absorption spectroscopy, Drs. J. B. Witten- berg and B. A. Wittenberg and Mrs. Lidija Kampa for the use of their extinction coefficients and spectra of pure MbOz and 21. for the use of their unpublished observations on the optimum conditions for preparation of long lived myoglobinIv, and Pro-

22 ’

fessor B. L. Horecker and Dr. A. Adler for helpful discussion. 23.

1.

2. 3.

VAN DEN BERGH, A. A. H., Deut. Arch. Klin. Med., 83, 86 25. (1905). 26.

MICHEL, H. O., Ph.D. thesis, Duke University, 1938. LEMBERG. R.. AND LEGGE. J. W.. Hematin comwounds and bile

pigmen&, Wiley-Interscience Publishers, Nkw York, 1949, pp. 490-499.

4.

5. 6. 7.

8. 9.

10. Il.

12.

13. 14. 15.

16.

NICHOL, A.W., HENDRY, I., MORELL, D.B., AND CLEZY, P.S., Biochim. Biophys. Acta, 156, 97 (1968).

HOPPE-SEYLER, F., ZbLMed. Wiss.,4,436 (1866). KEILIN, D., Proc. Roy. Sot. Ser. B Biol. Sci., 113,393 (1933). DRABKIN, D. L., AND AUSTIN, J. H., J. Biol. Chem., 112, 51

(1935-1936). MICHEL. H. O..J. Biol. Chem.. 126. 323 (1938'1 LEMBE~G, R., HOLDEN, H. F., ‘LEG&, J. ‘W., &VD LOCICWOOD,

W. H., Aust. J. Exp. Biol. Med. Sci., 20, 161 (1942). NICHOLLS, P., Biochem. J., 81, 374 (1961). * MORELL, D. B., CHANG, Y., AND CLEZY, P. S., Biochim. Bio-

phys. Acta, 136, 121 (1967). CLARKE, T. W., AND HURTLEY, W. H., J. Physiol. (London),

36, 62 (1907). MICHEL, H. O., J. Biol. Chem., 123, lxxxv (1938). GEORGE, P., AND IRVINE, D. H., Biochem. J., 60,596 (1955). HUGLI, T. E., AND GURD, F. R. N., J. Biol. Chem., 245, 1930

(1976).

17.

BLUMBERG, W.E., AND PEISACH, J.,in B. CHANCE, C.-P. LEE, AND T. YONETANI (Editors) 1 Structure and function of macro- molecules and membranes, Academic Press; New Yolk, 1971, in press.

KOLTHOFF, I. M., AND SANDELL, E. B., Textbook of quantitative inorganic analysis, Ed. 3, The Macmillan Company, New York, 1952, pp. 559-560.

18. FEHER, G., Bell System Tech. J., 26,449 (1957).

REFERENCES 24.

27.

28. KEILIN, D., AND H~RTRI%T, E. F., Biochem. J., 61, 153 (1955). 29. EDMUNDSON, A. B., Nature, 206, 883 (1965). 30. NIJVELD, H.‘A. W.,‘Rec. Tr&. Chim. @ays-kzs, 62,293 (1943). 31. DOROUGH, G. D., AND HUENNEI~ENS, F. M., J. Amer. Chem.

Sot., 74, 3974 (1952). 32. PLATT, J. R., Radiat. Biol., 3, 71 (1956). 33. BRILL, A., AND WILLIAMS, R. J. P., Biochem. J., 78,246 (1961). 34. WEISS, C., KOBAYASHI, H., AND GOUTERMAN. M.. J. Mol.

35.

36.

37. 38.

39.

40.

41. 42.

43.

HAPNER, K. D., BRADSH~W, R. A., HARTZELL, C. R., AND GURD, F. R. N., J. Biol. Chem., 243, 683 (1968).

PEISACH, J., AND BLUMBERG,W.E., inB. CHANCE, C.-P. LEE, AND T. YONET~NI (Editors), Structure and function of macro- molecules and membranes, Academic Press, New York, 1971, in press.

PEIUCH, J., BLUMBERG, W. E., OGAWA, S., RACHMILEWITZ, E. A.,AND OLTZIK, R., J. Biol. Chem., 246,3342 (1971).

HELCK~,G. A., INGRAM, D.J.E., ANDSLADE,E. F.,Proc.Roy. Sot. Ser. B BioZ. Sci.. 169.275 (19681.

HORECIQZR, B. L., J. BzIol. Chem:, 148: 173 (1943). HARTZELL, C. R., CLARIC, J. F., AND GURD, F. R. N.,J. BioZ.

Chem., 243, 697 (1968). GEORGE, P., AND HANANIA, G., Biochem. J., 52,517 (1952). GURD, F.R.N., FALK, K.-E., MALMSTR~M, B.G., AND VI~NN-

G%RD, T., J. BioZ. Chem., 242,5724 (1967). HANANIA, G. I. H., YEGHIAYAN, A., AND CAMERON, B. F.,

Biochem. J., 98, 189 (1966).

Spe&., i6, 415 (1965): I I

SMITH, D. W., AND WILLIAMS, R. J. P., Biochem. J., 110, 297 (1968).

DOROUGH, G. D., MILLER, J. R., AND HUENNEKENS, F. M., J. Amer. Chem. Sot., 73, 4315 (1951).

HILL, H. A. O., AND MORALLEE, K. G., J. Chem. Xoc., in press. PERRIN, D. D., Dissociation constants of organic bases in

aqueous solution, Butterworths, London, 1965, pp. 143, 153, 165.

PRYOR, W. A., Mechanisms of sulfur reactions, McGraw-Hill Book Company, Inc., New York, 1962, pp. 2733.

RACHMILEWITZ, E. A., PEISACH, J., AND BLUMBERG, W. E., J. BioZ. Chem., 246, 3356 (1971).

GRIFFITH, J. S., Nature, 180, 30 (1957). CAUGHII:Y, W. S., EBERSPAECHER, H., FUCHSMAN, W. H.,

MCCOY, S., AND ALBEN, J. O., Ann. N. Y. Acad. Sci.. 153. 722 (1969).

I

SHEARD, B., YAMANE, T., AND SIIULMAN, R.G., J.MoZ.Biol., 53, 35 (1970).

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Jay A. Berzofsky, J. Peisach and W. E. BlumbergSULFMYOGLOBIN AND ITS DERIVATIVES

Sulfheme Proteins: I. OPTICAL AND MAGNETIC PROPERTIES OF

1971, 246:3367-3377.J. Biol. Chem. 

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