1H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A

Biochimica etBiophysicaActa 952 (1988) 131-141 Elsevier

131

BBA33023

1 H . N M R h e m e r e s o n a n c e assignments by selective deuteration in low-spin c o m p l e x e s of ferric h e m o g l o b i n A

G e r d N. L a M a r a, T o m J u e a, K i y o s h i N a g a i b, K e v i n M. S m i t h a Y a s u h i k o Y a m a m o t o a, R o b e r t J. K a u t e n a V. T h a n a b a l a, K e v i n C. L a n g r y a, R a v i n d r a K. P a n d e y a a n d H i u - K w o n g L e u n g a a Department of Chemistry, University of California, Davis, CA (U.S.A.) and h MRC Laboratory of Molecular Biology, Hills Road, Cambridge (U.K.) (Received 16 June 1987) (Revised manuscript received 25 September 1987)

Key words: Hemoglobin; Ferric hemoglobin A; NMR, 1H-, 2H-; Low spin complex; (Selective deuteration)

The heme methyl and vinyl a-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole ~r bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.

Introduction

Nuclear magnetic resonance has played a key role in delineating some of the structural features that represent the ligated and unligated derivatives of the alternate quaternary states of human adult hemoglobin (Hb A) [1]. The ability to detect structural perturbations in the heme cavity which

Abbreviations: Hb A, human adult hemoglobin; Hb(III), ferric hemoglobin; Mb(III), ferric myoglobin; DDS, 2,2-dimethyl-2silapentane-5-sulfonate; NMR, nuclear magnetic resonance; a(IIl), isolated ferric c~-chain of Hb A; /~(III), isolated ferric B-chain of Hb A. Correspondence: G.N. La Mar, Department of Chemistry, University of California, Davis, CA 95616, U.S.A.

result from the quaternary allosteric transition is enhanced if the subunit being monitored is paramagnetic. This is due to the greatl~ expanded chemical-shift scale for the heme and beme-pocket residues in paramagnetic states relative to diamagnetic hemes and the exquisite sensitivity of these shifts to the slightest perturbations in the heine pocket [2-6]. In favorable cases, these shift changes accompanying a perturbation can be interpreted in terms of altered interaction in the proximal or distal pocket or at the heme periphery. Such a detailed understanding, however, has at its core the unambiguous assignment of the resonances being monitored. While low-spin ferric forms of many heme proteins are non-physiologically active, they have attracted much attention because the 1H-NMR reso-

0167-4838/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

132 nances are narrow, there is operative a single unpaired spin transfer mechanism, and the iron possesses magnetic anisotropy that imparts significant hyperfine shifts not only to the heme, but to many amino-acid side-chains in the heme pocket [2 17]. The latter amino-acid side-chain hyperfine shifts can yield valuable structural information on the heme pocket [7,8]. Early studies of such metcyano derivatives of myoglobin (Mb) and hemoglobin (Hb) proposed reasonable assignments based on relative peak area [9,10], the resonance position compared to assigned model compounds [11], or on the sensitivity of peak position on amino-acid substitutions [10,12]. Most prominent are sets of methyl signals which must originate from the heme, although possible assignments of vinyl and propionate signals have also been offered [9,13-15]. Assignment of sets of methyl peaks to individual subuntis has been made on the basis of comparison with either isolated chains [13,16] or with valency hybrids containing only one type of met-cyano subunit [15]. The additivity of the shift pattern from the respective chains to yield the spectrum of the tetramer, however, has been used both to assign the resonances and to conclude that the subunits structures are unaltered upon assembly of the tetramer [15,16]. Hence, an independent assignment of subunit peaks in a tetramer is highly desirable. Moreover, since the low-spin ferric heme resonances in hemoglobin have been shown to be sensitive to genetic origin of the chain [10,17], pH [14], and quaternary state of the tetramer [15], it is of interest to know the precise identity of the

heme side-chain (Fig. 1) responsible for the signal of interest. Isotope-labeled hemes reconstituted into lowspin ferric hemoprotein have been successfully utilized to assign heme resonance in myoglobin [18-20], various monomeric hemoglobins [21-23], cytochrome b 5 [24] and several peroxidases [25,26]. To date, the patterns of the methyl contact shifts in low-spin ferric hemes have been amenable to interpretation in terms of the nature of the axial interaction with the proximal His-F8 imidazole ring [5-7,27]. We have reported preliminary isotope-labeling studies on met-azido hemoglobin [28], where the location of the heme 5-methyl and 8-methyl resonances in both native and freshly reconstituted met-azido hemoglobin established that the latter holoprotein possesses a 1:1 disordered heine orientation with respect to a 180 ° rotation about the heme c~,y-meso axis (Fig. 1), and that such disorder persisted to a small degree in the/3 subunit of the native protein [29]. We report herein a more complete assignment of the heme resonances of low-spin ferric cyanide and azide complexes of the Hb A tetramer, including individual subunits, as well as in the met-cyano complexes of the isolated chains using the currently available isotope-labeled hemes. The present work confirms earlier assignment of sets of resonances to the respective subunits of hemoglobin [13,15,16], and provides the individual assignment of heme side-chains that allow comparison of the heme electronic/molecular structure to other hemoproteins in the low-spin ferric state. These assignments provide the necessary starting point for assignment of amino-acid side-chains using the nuclear Overhauser effect [8,30]. Materials and Methods

A

B

Fig. 1. Protohemin with labeling of side-chains. The rectangle represents the plane of the His-F8 imidazole as found in the X-ray structure of the native protein (A), and with the heme rotated by 180 o about the a , y - m e s o axis (B).

Packed human red blood cells were obtained from a local blood bank. The packed cells were diluted 1 : 1 in 1% NaC1 solution and washed three times by centrifugation at 500 × g for 20 min. The washed cells were lysed by dilution 1:1 with distilled water and freezing at - 3 0 °C under CO. After thawing, HbCO was separated from the lysed cells by centrifugation at 5000 × g for 20 min. HbO 2 was prepared by dissociation of CO from HbCO under strong illumination in the pres-

133

ence of oxygen. Met-aquo hemoglobin was prepared from HbO 2 by oxidation with potassium ferricyanide, followed by chromatography on a Sephadex G-25 column equilibrated with 20 mM Tris HC1 and 20 mM NaC1 (pH 6.9) to remove ferricyanide, ferrocyanide, and 1,3-diphosphoglycerate. The met-azido and met-cyano forms of Hb A were prepared by adding an excess of KCN or NaN 3. Isolated a- and B-chains were prepared by the method of Kilmartin et al. [31,32]. HbCO was reacted with p-mercuribenzoate and chromatographed on a DE-52 column ( W h a t m a n ) equilibrated with 10 mM potassium phosphate (pH 8.0). The a-chains were eluted with the column buffer. The B-chains were washed with column buffer containing 50 mM mercaptoethanol, and eluted with 60 mM potassium phosphate (pH 7.4). The a-chains were then incubated with 50 mM mercaptoethanol under CO for 30 min. The pH was adjusted to 6.6, and the chains were applied to a CM-23 column (Whatman), washed with 10 mM phosphate buffer containing 30 mM mercaptoethanol, and eluted with 33 mM Tris-HC1 (pH 8.5). CO was added to the air at the top of all columns. Apohemoglobin was prepared from isolated hemoglobin by the extraction procedure of Teale [33] followed by dialysis as described by La Mar et al. [28]. The separated apo-chains were prepared by the procedure of Yip et al. [34,35]. Apohemoglobin and its isolated apo-chains were reconstituted with native hemin or specifically deuterated hemins (see below), as described by La Mar et al. [28]. For the preparation of isotopelabeled hybrid tetrameric hemoglobin, the appropriate native and labeled chains were mixed

stoichiometrically just prior to determination of the N M R spectrum. The deuterated protohemins were prepared either by total synthesis [36] or by selective isotope exchange in native [37,38] or derivatized hemins [39,40]. In some labeled hemins, the degree of deuteration could be introduced highly selectively [36,40], and in these cases we refer to the hemin by the labeled position, i.e., [2,4-vinyl-a-2H]herrfin, which has only the two vinyl a-positions more than 99% deuterated [39] (see Table I). The labeled hemins which have less selective deuteration, we denote by using the primary deuteration site and insert it in quotes to indicate that other sites are also partially deuterated. One sample with desired 60-70% deuteration of methyl-1 and -3 also has secondary deuteration at the propioante and vinyl fl-positions [37], and hence is referred to as '[1,3methyl- 2H]hemin', in contrast to another similarly labeled sample in which secondary deuteration was avoided by use of newer synthetic techniques [40]. The labeled hemins are listed in Table I, together with the known degrees of deuteration at all positions, as determined by the 1H-NMR spectrum of the biscyano complex in methanol [41]. XH - N M R spectra were recorded at either 25 or 35°C on a Nicolet NT-360 FT N M R spectrometer operating in the quadrature mode at 360 MHz. Typical met-azido- or met-cyano-Hb A spectra were obtained by using a 12-kHz bandwidth, 8192 data points, a 6/~s 90 ° pulse, and 3000 scans. The water resonance was suppressed with a 300 ms presaturation decoupler pulse, and the signal-tonoise ratio was improved by apodization which introduced 10 to 30 Hz line broadening. Spectra designated 2H20 were from water samples containing greater than 90% deuteration of the solvent,

TABLE I D E U T E R I U M LABELED HEMINS Hemin designation

Ref.

Primary deuteration sites (%)

Secondary deuteration sites (%)

'[1,3-methyl- 2 H]Hemin' [1,3-methyl- 2 H]Hemin [1,5-methyl- 2 H]Hemin

37 40 36 [38] 39 39

3-methyl (71), 1-methyl (57) (90) 1-methyl (65), 5-methyl (90) (90) (100) (88)

6,7-propionate-fl- 2 H (55), 2,4-vinyl-fl- 2 H (58) none none

[6,7-propionate-fl-2H]Hemin [2,4-vinyl-a- 2 H]Hemin • [2,4-vinyl-fl- 2 H]Hemin'

nolle

none 2,4-vinyl-a- a H ( - 50) a,fl, y,&meso-2H2 (40-50)

134

while those designated H 2 0 contained 10% deuterium as internal reference. 2H-NMR spectra were recorded on a Nicolet NT-500 FT N M R spectrometer operating at 77.76 MHz. Met-azido hemoglobin spectra required a 30/~s 90 o pulse over a 7 kHz bandwidth with 4K data points and 15 000 scans. Chemical shifts are given in parts per million from 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), with : H 2 0 as internal reference.

F

ill

,

:1 I

li ill

i!

,),

,~

]J

f ~.

i

Results

solved portion of the 1H-NMR spectrum of Hb(III)CN in 2H20 is illustrated in Fig. 2A; the spectrum is the same as that reported previously [9,14,17], except for the present improved resolution and spectral sensitivity. Peaks A,A', B,B' have been attributed to methyls [9]. The upfield peaks D,D' arise from single protons, while E ' arises from two protons. All labeling is introduced equally into the a and fl subunit, so that only the heme positions can be identified. The use of '[2,4uinyl-/3-2H]hemin' (Fig. 2B) leads to a decrease of over half the intensity in the two-proton signal E'; hence E' arises from two vinyl-/3-Hs. Labeling solely the vinyl a-position (Fig. 2C) results in single proton intensity loss at 14.3 and 13.5 ppm, assigning peak C and C' to vinyl-c~-Hs. Reconstitution with '[1,3-methyl- 2H]hemin' leads to the metHbCN trace in Fig. 2D, which identifies B , B ' as either 1-methyl or 3-methyl, confirms E ' as vinyl-/3-H and suggests that D,D' arise from 6,7-propionate-/3-Hs. [1,5-methyl- 2H]Hemin_met_ HbCN yields the trace in Fig. 2E, which unequivocally establishes the origin of peaks B,B' as both 1-methyl, and peaks A,A' as both 5-methyl. Reconstitution with [6,7-propionate-/3-ZH]henfin, Fig. 2F, unequivocally confirms that peaks D,D' arise from propionate-/3-Hs. The slight variability of the shape of the composite peak(s) near 13-14 and - 3 ppm is due to the strong pH sensitivity [14] of the positions of particularly these resonances. Isotope-labeled hybrids. In this section, we consider the Hb(III)CN tetramer when the isotope labeling has been carried out in only one of the two types of subunit. Incorporation of '[1,3methyl-2H]hemin ' solely into the c~ subunit of

~

J,

Met-cyano hemoglobin Isotope-labeled hemoglobin tetramer. The re-

%,

J

I"!1,

\

!%1

,L J 2~

22

20

18

15

1N

I2

PFM

_

Fig. 2. Hyperfine shifted region of the 360 MHz ]H-NMR spectra of human adult Hb(III)CN and isotope-labeled derivatives in H20 or 2H20 , 0.2 M NaCl, 0.1 M Bistris at 35°C and a nominal pH 6.5. (A) Native protein in 2H20; (B) '[2,4-vinylfl-2H]hemin'-Hb(IlI)CN in 2H20; (C) [2,4-vinyl-a-2H]hemin Hb(III)CN in 2H20; (D) '[1,3-methyl-2H]hemin'-Hb(III)CN in 2H20; (E) [1,5-methyl-2H]hemin-Hb(III)CN in 2H20; (F) [6,7-propionate-B-2H]hemin-Hb(lII)CN in H20. The intensity of the upfield portions is reduced by 0.7 compared to the downfield portion. Primary positions of deuteration are indicated by long arrows, and secondary positions of deuteration are shown by short arrows.

hemoglobin results in the N M R trace in Fig. 3B, which is compared to the native protein in Fig. 3A. The 1-methyl origin of peak B is confirmed, as is the 6 or 7-/3-H origin for peak D, and it is established that both B and D arise from the a subunit. Conversely, since peak E' suffered no intensity loss whatsoever, both of the constituent vinyl-/3-H peaks must originate in the/3 subunit. Reconstitution of solely the a subunit with [2,4vinyl-ZH]hemin yields the N M R trace in Fig. 3C; here the loss of a peak is less obvious, although the area loss, particularly compared to the trace in Fig. 3B (which is at the same pH as trace C), appears to be at 14 ppm, indicating peak C as an c~ subunit vinyl-c~-H (see below). Reconstituting '[1,3-methyl-2H]hemin ' solely into the /3 subunit

135

D

,

D

~

2~i

22

20

~

.

1B

, B B'~ CA

A

16

12 pPfl

1½

'

-S

Fig. 3. Hyperfine-shifted region of the 360 mHz I H - N M R spectra of human adult Hb(III)CN and isotope-labeled hybrids in 2H20, 0.2 M NaCI, 0.1 M Bistris, at 3 5 ° C and nominal pH 6.5. (A) Native protein, [a(III)CN]2[fl(III)CN]2; (B) '[1,3methyl-2H]hemin'-a(III)CN]2[fl(III)CN]2; (C) [2,4-vinyl-a2H]hemin-a(III)CN]2[fl(III)CN]2; (D) [a(II1)CN]2'[1,3methyl-2H]herrfin'-fl(III)CN]2. The intensity of the upfield portion is reduced by 0.7 relative to the downfield portion. Positions of primary and secondary deuteration (Table I) are designated by long and short arrows, respectively.

of metHbCN leads to the decrease of intensity of peaks B', D' and E' in Fig. 3D, confirming that these originate from the fl subunit 1-methyl, two propionate-fl-Hs, and two vinyl-fl-Hs, respectively. The chemical shifts and assignments are listed in Table II. Isolated met-cyano chains. The reference spectrum for the a(III)-CN, complex is shown in Fig. 4A, which compares well with previously reported

A

1

25

. . . .

B

I

. . . .

20

I

15

. . . . .

PPN

Fig. 4. Hyperfine-shifted portions of the 360 MHz 1H-NMR spectra of isolated a(III)CN chains and isotope-labeled derivatives, in 2H20, 0.2 M NaC1, 0.1 M Bistris, 2 5 ° C and a nominal pH 6.5. (A) Native a(III)CN; (B) [2,4-vinyl-a2H]hemin-a(IlI)CN; (C)'[1,3-methyl-2H]hemin'-a(llI)CN; (D) [1,5-methyl-2H]hemin-a(IIl)CN. Intensities of the upfield portions are reduced by 0.5 comapred to downfield portions. Positions of primary and secondary deuteration (Table I) are indicated by long and short arrows, respectively.

spectra [14] that have attributed peaks A and B to methyls and C and D to single protons, and the peak at -3.2 ppm to a composite of two methyl peaks. Reconstituting the a chain with [2,4-vinyl-

TABLE II CHEMICAL SHIFTS OF ASSIGNED HEME RESONANCES IN MET-CYANO Hb A AND ITS SEPARATED CHAINS a Assignment b

Hb(III)CN

(peak labels)

a subunit

fl subunit

5-Methyl (A,A') 1-Methyl (B,B') Vinyl-a-H (C,C') Propionate-fl-H (D,D') Vinyl-fl-H (E,E')

22.3 16.4 14.4 - 1.5 _ c

22.2 15.5 13.6 - 1.8 -4.7

a Shifts in ppm from DDS at 25 o C and pH 6.5. b Assignments as in Figs. 2 and 3. c Not detected.

i

-5

a(III)-CN

fl(III)-CN

22.3 16.0 14.1 - 2.0 - c

21.1 15.4 13.5 - 1.9 - 4 . 4 , 4.6

136

a-2 H]hemin yields the trace in Fig. 4B, and establishes that C arises from a single vinyl-a-H. Incorporating '[1,3-methyl-2H]hemin ' into a(III)-CN results in trace 4C, which dictates that B arises from either 1-methyl or 3-methyl, and D from a propionate 6,7-/3-H or possibly a vinyl-B-H; the degree of deuteration (Table I) of 6,7-fl-H and 2,4-fl-H is comparable [37]. Comparison to the tetramer favors its former assignment. The use of [1,5-methyl-2H]hemin in Fig. 4D, in conjunction with Fig. 4C, clearly establishes that A = 5-methyl and B = 1-methyl. Chemical shifts for assigned peaks are included in Table I. The reference trace for/~(III)-CN is illustrated in Fig. 5A; relative areas indicate that A' and B' arise from methyls, C' from a two-proton signal (which each split into two signals at extreme temperatures), and D', E' and F' from single proton peaks. The relative instability of the fl-chain allowed fewer successful reconstitutions. Incorporation of [2,4-uinyl-a-2H]hemin yielded a sample with the trace shown in Fig. 5B. The decrease in intensity of peak C' by one-half establishes the location of a single vinyl-a-H peak. Reconstitution with '[1,3-methyl-2H]hemin' leads to a trace shown in Fig. 5C. Thus peak B' is the 1-methyl or 3-methyl, and peaks D', E' and F' arise from

1

A'

25

I 2O

. . . .

I 15

PFId

0

-5

Fig. 5. Hyperfine-shifted portions of the 360 MHz 1H-NMR spectra of isolated fl(III)CN chains and isotope-labeled derivatives, in H20, 0.2 M NaC1, 0.1 M Bistris, at 2 5 ° C and a nominal pH 6.5. (A) Native fl(III)CN; (B) [2,4-vinyl-aZH]hemin-fl(III)CN; (C)'[1,3-methyl-2H]hemin'-fl(III)CN. Intensities of the upfield and downfield portions are to the same scale. Positions of primary and secondary deuteration (Table I) are indicated by long and short arrows, respectively.

either 6,7-propionate-fl-H or 2,4-vinyl-fl-Hs. It is noted that the 60% deuteration of 1-methyl in trace C indicates that there is probably a single proton peak under the 1-methyl. The chemical shifts are included in Table II.

Met-azido hemoglobin Isotope-labeled tetramer. The resolved low-field portion of the ]H-NMR spectra of both native and freshly reconstituted Hb(III)N 3 are illustrated in A and C, respectively, of Fig. 6. The native spectrum consists of three pairs of prominent peaks attributed to heme methyls. The assignments of methyls A, B and C to the a, and A', B' and C' to the fl subunit has already been established unequivocally [29,42]. Reconstitution with [1,5methyl-2H]hemin leads to the trace for the equilibrated protein as shown in Fig. 6B. Peaks A,A' had been shown previously to both arise from the 5-methyl; A from the a and A' from the fl subunit, respectively [28,29]. Similarly, peaks B,B' have been unambiguously attributed to the a and fl subunits, respectively [28,29]. Trace 6B establishes that both B and B' arise from 1-methyl. The peaks C,C' had been previously shown to arise from 8-methyl from the a and fl subunits, respectively [28,29]. This assigns all of the resolved peaks to both individual subunits and specific heme methyl in the native protein. The location of the last of the heme methyls (3-methyl), which must resonate in the intense diamagnetic envelope, was approached by utilizing 2H-NMR of the labeled [1,3-methyl-2 H]hemin. Narrow 2H-NMR lines can be observed due to rapid methyl rotation [43,44], while other residual deuteration of immobile vinyl and propionate groups leads to extremely broad, and hence undetectable, lines. Moreover, we pursue here the location of the remaining heme methyl lines in both the native as well as the freshly reconstituted 1 : 1 disordered protein [28,29] (reference trace at 35 °C in C of Fig. 6. In addition to the assigned methyl peaks of the native heme orientation, peaks A, B, C, A', B' and C', we note methyl peaks a,a' and b,b', and single proton peaks e,e', which have previously been assigned to the 8-methyl, 5-methyl and 4-a-H of the a and fl subunits with the reversed-heme orientation, respectively [28]. The 1H-NMR trace of a freshly reconstituted approx.

137

1:1

disordered Hb(III)N 3 sample using [1,3-

rnethyl-2H]hemSn is illustrated in D of Fig. 6. F

B B'

B'

'

I

30

'

'

'

'

I

20

C C'

'

""

'

'

I

tO

0

-10

Fig. 6. Hyperfine-shifted regions of NMR spectra of native and reconstituted Hb(III)N 3 and isotope-labeled derivatives, in 2H20 or H20, 0.2 M NaC1, 0.1 M Bistris. (A) 360 MHz 1H-NMR spectrum of native Hb(III)N 3 in 2H20 at 25 ° C (pH 6.5). Peaks A-C, and A ' - C ' arise from the a and fl subunits, respectively, for the native heme orientations (Fig. 1A); (B) 360 MHz 1H-NMR spectrum of [1,5-methyl-2H]hemin-Hb(III)N3 in 2H20 at 25°C (pH 6.5); (C) 360 MHz 1H-NMR spectrum of freshly reconstituted Hb(III)N 3 at 35 o C, (pH 8.3). The new peaks a-c, e and a ' - e ' , e ' arise from the a and fl subunits, respectively, for the reversed-heme orientation (Fig. 1B); (D) 360 MHz 1H-NMR spectrum of freshly reconstituted [1,3methyl-2H]hemin-Hb(III)N3 at 35°C in H20 (pH 8.1). Both pairs of peaks B,B', and e,c' are reduced in intensity; (E) 76.7 MHz 2H-NMR spectrum in H20 at 35°C (pH 8.1) of the same freshly reconstituted [1,3-methyl-2H]hemin-Hb(III)N3 used for D above; (F) 76.7 MHz 2H-NMR spectrum in H20 at 35°C (pH 7.8) of the sample from trace D after allowing appreciable equilibration of the heme; note increased inten-

Missing in this trace are the two sharp resonances, e,e', at 13.2 and 11.5 ppm, respectively, which are also missing in the native protein spectrum (not shown at the same temperature). Thus e,e' arise from either the 1-methyl or 3-methyl of the heme with the reversed-heme orientation as in B of Fig. 1. The 2H-NMR trace of approx. 1 : 1 disordered Hb(III)N 3 freshly reconstituted with [1,3-methyl2H]hemin is illustrated in E of Fig. 6. As expected, two peaks, B,B', correspond exactly in shift to the assigned aH-NMR 1-methyl peaks B,B' for the native protein (Fig. 6C). The second set of peaks, c,c', exhibit the same chemical shift as the methyl peaks, c,c', assigned in traces C and D above. In agreement with their assignment to the metastable orientation (B in Fig. 1), peaks c,c' rapidly lose intensity as the sample in Fig. 6E is allowed to equilibrate (Fig. 6F). The 2H-NMR trace of the 1:1 disordered Hb(III)N 3 (Fig. 6E) exhibits two more clearly resolved resonances which can be attributed to methyls, peaks D and D'. Since their intensity relative to peaks B,B' remain the same when the sample equilibrates, they must belong to the native-heme orientation (A in Fig. 1), and hence must be the 3-methyl peaks for the two subunits. The remaining pair of methyl peaks for the heme orientation as in Fig. 1B are suggested to resonate at the low-field shoulder of the residual water peak (peak d as the shoulder, peak d' under the solvent resonance). The shoulder is clearly lost on allowing the sample to equilibrate (Fig. 6F). Thus, all heme methyls for both heme orientations have been located. However, the available 2HN M R data do not allow direct assignment to individual subunits of the unresolved methyls. The shifts of assigned peaks are given in Table III. Isotope-labeled chains. The trace of isolated a(III)-N 3 is illustrated in Fig. 7A, which consists of three dominant apparent methyl peaks, A, B and C, a two-proton peak D, and a composite peak upfield consisting of probably two methyls. Labeling with [2,4-vinyl-a-ZH]hemin (Fig. 7B) sities of B,B' and D,D' at the expense of c,c' and d,d'. Positions of lost 1H-NMR intensity due to methyl deuteration are indicated by arrows.

138 T A B L E Ill C H E M I C A L SHIFTS OF A S S I G N E D H E M E RESONANCES IN M E T - A Z I D O Hb A A N D ITS S E P A R A T E D C H A I N S ~ Assignments b

Hb(III)N 3

(peak labels)

a subunit

a(ili)_N 3 /~ subunit

Native-heme orientation (Fig. 1A) 5-Methyl (A,A') 1-Methyl (B,B') 8-Methyl ( C , C ' ) 3-Methyl ( D , D ' ) Vinyl-a-H (E,E')

27.8 21.7 15.5 1.2 _ c

26.2 20.5 14.3 - 0.6 _ b

28.1 22.3 15.3 14.1

Reversed-heme orientation (Fig. 1B) 8-Methyl (a,a') 5-Methyl (b,b') 3-Methyl (e,e') 1-Methyl (d,d') 4-Vinyl-a-H (e,e')

27.8 17.1 13.2 --- 7 21.7

26.2 15.5 11.4 = 5 (under 2HHO) 18.3

" Shifts in p p m from DDS, at 25 ° C and pH 6.5. b Assignments as in Figs. 6 and 7. c Not detected.

locates one vinyl-a-H under peak D. Reconstitution with '[1,3-methyl-2H]hemin ' (Fig. 7C) establishes that peak B originates in either 1-methyl or 3-methyl, while the similar use of [1,5-methyl2H]hemin (Fig. 7D) clearly identifies peak A as 5-methyl and B as 1-methyl. N o attempts were made to obtain N M R spectra of labeled isolated met-azido /3-chains. The relevant shifts for assigned peaks are included in Table III. Discussion

Met-cyano hemoglobin assignment

35

30

25

20

15

PPM

-5

Fig. 7. Hyperfine-shifted region of the 360 M H z 1 H - N M R spectra of isolated a ( l l I ) N 3 chains and isotope-labeled derivatives, in 2H20, 0.2 M NaC1, 0.1 M Bistris, 2 5 ° C and a nominal pH 6.5. (A) Native a(IlI)N3; (B) [2,4-vinyl-a2H]hemin-a(lll)N3; (C)"[1,3-methyl-2H]hemin'-a(lll)N3; (D) [1,5-methyl-2H]hemin-a(Ill)N3 . The intensity of the upfield portion is reduced by 0.7 compared to the downfield portion. Position of deuteration is indicated by arrows.

The labeling of Hb(III)-CN and hybrids establishes that the c~ subunit gives rise to peak B (1-methyl), C (vinyl-a-H), D (propionate-/3-H), and A or A' (5-methyl), while the/3 subunit yields A' or A (5-methyl), B' (1-methyl), C' (vinyl-a-H), D' (propionate-/3-H) and E' (two vinyl-/3-Hs). The unambiguous assignment of B and B' to the and /3 subunits, respectively, confirms the earlier proposal based solely on valency hybrids possessing a single met-cyano subunit [15]. The assignment of A and A' to individual subunits is not established here, but it is likely that A is from the c~ subunit, as previously proposed [15]. The 3-

139 methyl and 8-methyl signals for both subunits are obscured by the intense diamagnetic envelope 0-10 ppm. However, since the contact shift for 1-methyl is much larger than for 3-methyl in both subunits, we expect the 2-vinyl group to exhibit larger contact shifts than the 4-vinyl groups, as observed in other related met-cyano myoglobin complexes [45], such that C,C' must arise from 2-a-H, and E ' from the two 2-/3-Hs. The two/3-Hs from the a subunit may be under the intense composite peak at - 2 . 7 ppm. At this time, there is no basis for distinguishing between the 6- and 7-propionate groups as the origin for peaks D,D'. The remaining low-field resonances in the window, 12-14 ppm, and under B and B' must arise from either propionate-a-Hs or amino-acid side-chain protons. A synthetic route for efficient incorporation of deuterium into propionate a-positions remains to be developed. It was noted earlier that the resonance position of methyl peak A' of Hb(III)CN is sensitive to sequence differences among Hb A, Hb F and horse Hb, and the difference was suggested to be due to variations in the amino acid at position 70 [10]. The current assignment shows that, in fact, this sensitive resonance arises from the 5-methyl which is in contact with the variable residue in these three proteins [46]. The labeling of a(III)-CN similarly identifies A as 5-methyl, B as 1-methyl and C as a vinyl-a-H, which we can attribute to the 2-a-H. The single proton peak, D, could arise from either a vinyl-/3-H or a propionate-fl-H, since these two positions are comparably labeled. However, due to the close correspondence of shifts for given functional groups in the isolated chains and for that subunit in the tetramer (see below), we favor D as arising from a propionate-/3-H. For/3(III)-CN, we can only confirm that B' is due to 1-methyl or 3-methyl, and identify C' as a vinyl-a-H (from the same pyrrole on which the methyl resides which yields A'). Analogy to Hb(III)-CN dictates that B' is 1-methyl and A' is 5-methyl, such that C' is due to 2-a-H. The peaks D', E ' and F' could arise from either propionatefl-H or vinyl-fl-H. Again, because of the very similar shifts in the isolated chain and the fl subunit in Hb(III)-CN (see below), we assign D' to the propionate, and E ' and F' to the two 2-vinyl-/3-Hs.

It is noteworthy that not only do the methyl shifts in the two chains correspond closely in shift to that of the same subunit in the tetramer, but that this is true for the 2-a-Hs, the propionate-/3-Hs and the vinyl 2-/3-Hs as well (see Table I). Thus, it appears that there is indeed very little perturbation on the heme pocket structure upon assembling the R-state Hb(III)-CN from the respective separated chains [13-16].

Met-azido hemoglobin assignments For the tetramer, only six heme methyls are resolved from the intense dismagnetic envelope in the native protein. The assignments of three peaks to each subunit, A, B, and C from the a, and A', B' and C' from the /3 subunit has been established unequivocally [28,29,42] by taking advantage of the known difference between the subunits in their affinity for azide in the met-aquo state [47], and the rate of autoxidation in the reduced oxy state [48]. The present assignment of B,B' to 1-methyls completes the assignment for the native tetramer (Table III). 2H-NMR locates the 3-methyl, peaks D,D' (Fig. 6D and E). While our data do not distinguish between the subunits, we tentatively assign D and D ' to the a and /3 subunits, respectively, on the basis that for each of the other three methyl groups, the a subunit appeared on the low-field side of the/3 subunit peak. The freshly reconstituted Hb(III)-N 3 exhibits twice the number of resolved resonances due to 1 : 1 population of the two heme orientations [28,29], as depicted in Fig. 1. The six resolved peaks for the reversed-heme orientation have been assigned to the 8-methyl (a,a'), 4-a-H (e,e') and 5-methyl (b,b') with a and/3 subunits, respectively [28]. The labeling with [1,3-methyl-ZH]hemin identifies peak e,e' as 1-methyl or 3-methyl. Based on the larger contact shift for the 4-vinyl than 2-vinyl group, we attribute e,e' to 3-methyl. The 1-methyl peaks must be d,d' (under residual 1HHO). Again our ZH-NMR data do not distinguish between subunits, but on the basis of the relative shifts of other peaks for the reversed-heme orientation, we assign e and d to the a, and e' and d' to the fl subunit. The complete assigned shifts for the reversed-heine orientation are also included in Table III. It is noted that the linewidth for the 2H signals

140 for methyl B,B' is approx. 160 Hz. Assuming rapid internal methyl rotations, the dominant quadrupolar relaxation mechanism and the known methyl quadrupole coupling constant [43,44] yield a correlation time for overall tetramer reorientation of 35 ns at 25 ° C. This agrees well with other estimates for the hemoglobin tetramer [49], and suggests that such ZH-NMR methyl linewidths may serve as a useful probe of the extent of the association of subunits for heme proteins. H e i n e electronic s t r u c t u r e

Both Hb(III)-CN and Hb(III)-N 3 are predominantly low-spin, with the latter exhibiting a small degree of high-spin character [50]. The pattern of the heine methyl shifts is the same in both types of complex, and is maintained in the separated chains, where determined. The heme methyl contact shift patterns in low-spin ferric heroes have been interpreted on the basis of the protein-induced rhombic perturbation which raises the orbital degeneracy of the iron spin-containing d orbitals [5-7,18-28]. The dominant protein influence has been proposed to originate from the proximal His-F8 imidazole-iron ~r bonding [7,27]. The spread of the heme methyl contact shifts reflects the location of the rhombic perturbation. For the His-F8 imidazole rt plane projection near a N - F e - N vector (Fig. 1), the spread in shifts is maximum and the methyls on the pyrroles perpendicular to the His-F8 imidazole plane exhibit strongly downfield-shifted peaks, while the other two methyls exhibit negligible contact shifts and hence usually resonate within the intense diamagnetic envelope. As the His-F8 imidazole plane rotates away from a N - F e - N axis, the spread of the methyl shift decreases until the shifts become essentially degenerate for an orientation passing through opposite m e s o positions. The known HisF8 orientation in Hb A is as depicted in Fig. 1A [46], and the low field 1-methyl and 5-methyl shifts are completely consistent with this, as found also for myoglobin [18,19,51]. This lends support to the use of the heme methyl contact shift pattern to determine the orientation of the heme in the heme pocket, or to detect heme rotational disorder in other low-spin ferric hemoproteins [18-28]. The heme methyl shift change for the cyano-met a subunits in the valency hybrid of Hb A both

exhibit an upfield bias upon oxygenating the ferrous subunit [15]. Such ligation has been proposed to trigger the R ~ T transition. On the basis of the above model for the contact shifts, such a change suggests a small rotation of the His-F8 imidazole plane away from the N ( I I ) - F e - N ( I V ) axis. X-ray structural data on the valency hybrids in question are, unfortunately, not available. The very similar hyperfine shift pattern for the two subunits in Hb(III)-CN or Hb(III)N 3 reflect their close structural similarity. This near-pairing of the shifts of the same functional group allows for direct comparison of selective environmental changes in subunits based on systematic perturbations [10]. This is in contrast to deoxy hemoglobin, where the similarly hyperfine-shifted heme methyls from the two subunits have their origin in different functional groups (La Mar, G.N., Jue, T., Nagai, K., Smith, K.M. and Langry, K.C., unpublished data).

Conclusions The prominent hyperfine-shifted resonances of Hb(III)-CN and Hb(III)-N 3 have been assigned to both subunits and to individual positions on the heme skeleton. The assignments and chemical shifts for resonances are essentially the same in separated chains and the subunits of the tetramer, confirming that the structures of the heme cavity are largely unaltered upon the assembly of the R-state tetramer from the separated chains. The patterns of heme methyl contact shifts for subunits of the tetramer or separated chains are consistent with being determined by the rhombic perturbations resulting from the ~r bonding of the proximal histidyl imidazole.

Acknowledgement This research was supported by grants from the National Institutes of Health, HL-16087, HL22252.

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