Paramagnetic 'H NMR Saturation Transfer Study of Ligand Exchange in Iron(II1) Myoglobins

Yasuhiko Yamamoto* and Yoshio Inoue

Tomohiko Suzuki

Department of Riomolecular Fngineermg, Tokyo Institute of Technology, 4259 Nagatsuta Midnri-ku, Yoknhama 227 Japan

Department of Biology, Faculty of Science, Kochl Univenity. Kochl 780, Japan

'H NMR saturation transfer experiments were successfully used in connecting iron(II1) high- and low-spin forms of equine, the mollusc Dolabella auricularia and the shark Mustelus japonicus myoglobins. With the known signal assignments in the high-spin form, a haem peripheral proton resonances in met-azido and met-imidazole complexes of the myoglobins have been straightforwardly assigned via the saturation transfer connectivities. Analysis of the extent of the saturation transfer provided the kinetics of the ligand exchange.

KEY WORDS NMR Paramagnetic myglobin Saturation transfer

I N T R O D U C T I O N

'H NMR presents potentially the most powerful spec- troscopic method for elucidating detailed structures of haemoproteins in all oxidation and spin states.'92 In the paramagnetic haemoproteins, signals arising from the prosthetic haem peripheral protons (see Fig. 1) are resolved well outside the poorly resolved diamagnetic envelope in the chemical shift region 0-10 ppm and provides a particularly sensitive probe for the haem environments. Since the hyperfine shift is quantitatively interpretable in terms of the interaction between the nucleus and the unpaired electron@) of the haem i r ~ n , ~ . ~ the molecular and electronic structure of the haem active site can be directly inferred from the analysis of the resonance shifts.

The analysis of NMR spectra first demands unam- biguous signal assignments. During the initial effort for the specific assignments of the haem peripheral proton

H

Figure 1. Structure and numbering system of the haemi.

* Author to whom correspondence should be addressed.

0749-1 581/93/SIO008--09 $09.50 $;> 1993 by John Wiley & Sons, Ltd.

resonances in the paramagnetic haemoprotein, reconsti- tution with isotope-labelled haem has been mainly used and has led to some important assignment^.^ ' Later, the nuclear Overhauser effect (NOE),*-I3 together with paramagnetic reIaxation,l4*' 2D NMR'6-20 and genetic mutants,21 has been successfully utilized for unambiguous assignments of the resonances for both the haem and amino acid residues in the haem active site. Further, saturation transfer (ST) has also provided a unique tool for assigning the haem resonances.z2 24

The ST experiments are uqeful for relating resonances which are connected by a dynamic exchange process with a suitable time scale. Hence, if the assignments are known in one form, the ST connectivity identifies the same resonance in the other form.

The iron(II1) high-spin form of myoglobin (metMb) possesses either pentacoordinated haem iron2 1 9 2 5 2 7 or hexacoordinated haem iron2' with H,O as an Fe- bound ligand and binds external Iigands such as CN , N 3 - , O H - and imidazole. The ligand binding of metMb results in a change of the total spin of the haem iron from iron(II1) high-spin (S = 5/2) to iron(II1) low- spin (S = 1/2). Since the hyperfine shift is proportional to S(S + 1)/3,3 there is a large difference in the reson- ance frequency between the two different spin states of Mb. With the amount of ligand insufficient to convert metMb completely into iron(II1) low-spin Mb, the signals from both forms are often observed separately. In the spectrum of metMb, a large scalar interaction leads to the resolution of most haem peripheral proton signals and the previous assignments in various

d emonstrated that the haem methyl hyperfine shift pattern in metMb is independent of the protein. Hence the haem methyl proton signals in iron(II1) low-spin Mb can be straightforwardly assigned via the ST connectivities upon the saturation of the cor- responding resonances in metMb.

We present here the results of an ST study of equine Mb and Mbs from the mollusc &k&dQ

Receiwd 25 May I993 Accepted (reuiwd) I S Auyzist 1993

PARAMAGNETIC 'H NMR SATURATION TRANSFER STUDY s9

auricularia' 5.24*27*29 and the shark Mustelus japon- icus," which demonstrate the potential applicability of the ST experiments of assigning the haem resonances in met-azido Mb (metMbN, - ) and met-imidazole Mb[metMb(Im)]. In Dolabella metMb, the effect of N, - exchange on the intrinsic spin-lattice relaxation time (Tpr) of a haem methyl proton resonance is inter- preted in terms of the kinetics of the ligand exchange. The haem methyl hyperfine shift pattern in metMbN, ~ s yields valuable insight into the haem electronic struc- ture.

PRINCIPLE

The ligation of metMb can be represented by the fol- lowing reaction:

km

m e t M b + L - metMbL-

where k,, and koff are the association and dissociation rate constants, respectively. The interconversion between the two forms of Mb on a time scale compara- ble to or faster than the intrinsic relaxation times of nuclei leads to establishing ST connectivities between resonances. If assignments are known in one form of Mb, the connectivities identify the same resonance in the other form.

On saturation of a signal in one form the equilibrium intensity, Zeq, of the same signal in the other form is reduced to I,, . The ST factor (Zst/leq) is expressed as3'

(1) where TP is the intrinsic spin-lattice relaxation time of the resonance and z is the lifetime of the species, the signal intensity of which is analysed. Hence, for the observation of ST, i.e. I s J Z e q < 1, z must be comparable to, or preferably smaller than Tyr.

In the present case, TYr values for haem methyl proton resonances in metMb and metMbL- are <10 and -20 ms (see below), respectively. Hence, if z is equivalent for both forms, the ST connectivity is more easily detected on the resonance of metMbL- on satu- ration of the resonance of metMb, provided that the radiofrequency irradiation power of the instrument is strong enough to saturate completely a desired signal with its TFtr of < 10 ms.

koff

ZsJZeq = (T';"") - I/[( T y ) - + z - '1

EXPERIMENTAL

Sample preparation

Dolabella Mb was extracted from its triturative stomach and purified according to the method described p r e v i o ~ s l y . ~ ~ Mustelus Mb was isolated from red muscle and purified as reported previo~sly.~' The Mb samples were oxidized by the addition of a fivefold molar excess of potassium hexacyano-ferrate(II1) (Sigma Chemical). metMb was separated from the residual reagents with a Sephadex G-50 (Sigma Chemical) column equilibrated with 10 mM Bis-Tris buffer (Sigma Chemical) of pH 6.8. Equine Mb was purchased from Sigma Chemical and used without further purification. Sodium azide and imidazole were also obtained from Sigma Chemical. Mb solution was concen-

trated to about 0.5 mM and then solvent was exchanged with D,O in an Amicon ultrafiltration cell. pD was measured using a Toko Model TP-10 pH meter with a Toko CE103C electrode. The isotope effect was not considered to correct the pD value.

NMR spectroscopy

'H NMR spectra were recorded using a JEOL GSX-270 Fourier transform NMR spectrometer oper- ating at a 'H frequency of 270 MHz. A typical spectrum consisted of 2K transients with 16K data points over 70 kHz band width and a 9.5 ps 90" pulse. The residual water resonance was suppressed with a 40 ms presatu- ration decoupler pulse. Intrinsic spin-lattice relaxation times ( TFtr) were measured using the saturation- recovery method with a selective saturation pulse.24 ST experiments were carried out by selectively saturating a desired peak for 40 ms. The spectra resulted from the ST experiments were presented in the form of a differ- ence spectrum. The ST factor ( I , JIeJ was determined by integrating the peak area. The signal-to-noise ratio of the spectra was improved by apodization, which intro- duced 50 Hz line broadening. 2D exchange spectra were recorded using the standard pulse sequence with mixing times of 5 and 10 ms for Dolabellu and equine metMbs and Mustelirs metMb, respectively; 256 free induction decays were acquired with 1K data points and a spec- tral width of 70 kHz. The time-domain data matrix was expanded to 1K x 512 by zero-filling. A phase-shifted sine-squared function was used to apodize the spectrum in both dimensions and the spectra were presented in the absolute value mode. The chemical shifts are given relative to sodium 2,2-dimethyl-2-silapentane-5-sul- phonate with the residual H D O as internal reference.

RESULTS

N,- titration of equine metMb

The downfield hyperfine shifted portion of the 270 MHz 'H NMR spectrum of 0.5 mM equine Mb at pD 7.56 and 35°C is illustrated in Fig. 2(A). Signals resolved at >30 ppm arise from the haem peripheral protons of metMb. Four haem methyl proton signals have been assignedz6 and are indicated with the spectrum. With the addition of N3-, the intensity of the signals at <40 ppm for metMbN; increases at the expense of those for metMb, as shown in Fig. 2(B) and (C). The line broadening observed for the signals of metMb in Fig. 2(B) is attributed to the exchange reaction between metMb and met-hydroxyl-Mb at the pH value used, 8 . 2 3 . j ' ~ ~ ~ The difference in the total spin between metMb (S = 5/2) and metMbN,- (mainly S = 1/2)34 results in a large separation of resonances arising from the two forms, which allows independent observation of their signals. The difference in the resonance frequency of > 6 kHz between the two forms dictates that the N, - exchange rate is <0.6 x lo3 s-'. From the similarity in the spectral patterns between equine and sperm whale metMbN,- s," the haem methyl proton signals of equine metMbN, - are tentatively assigned as those of sperm whale metMbN,-. The [metMbN, -]/[metMb]

s10 Y. YAMAMOTO Y. INOUE AND T. SUZUKI

I metMbNg- 3l

160 80 50

Figure 2. Downfield hyperfine shifted portions of the 270 MHz 'H NMR spectra of 0.5 mM equine metMb at pD 7.56 and 35"C(A) and in the presence of 0.25 mM N,- at pD 8.23(6) and 5 mM N3- at pD 8.41 (C). Signals from metMbN,- are observed at <32 ppm and, with the addition of N,-, the intensity of these signals increases at the expense of those for metMb. The signal assignments of the haem methyl proton resonances of metMbZ6 and metMbN; are indicated in (A) and (C), respectively.

value of 0.54 in Fig. 2(B) is reasonably consistent with the reported k,, and koff values (at 20 "C, in 0.1 M phos- phate buffer, pH 7.0)35 of 2.6 x lo3 1 mol-' s-' and 3.8 x lo-' s- ', respectively.

Signal assignments in equine metMbN,- via ST connectivities

The ST difference spectra of the mixture of equine metMb and metMbN, - ([metMbN,-.]/[metMb] = 0.54) at pD 8.23 and 35°C are illustrated in Fig. 3. On saturation of the haem methyl proton resonances of metMb, a smaller but clear ST was detected on the cor- responding methyl proton resonance of metMbN, -. Therefore, the haem methyl proton signals of metMbN, - can be assigned and these ST connectivities confirm the tentative haem methyl proton signal assign- ments in equine metMbN,-. These results clearly demonstrate the potential applicability of the ST method for the signal assignments in paramagnetic Mb.

Signal assignments in Dolabella metMbN,- via ST connectivities

The ST experiments were applied to the mixture of Dolabetla metMb and metMbN,- ([metMbN,-]/ [metMb] = 1.2) at pD 7.24 and 30°C and the results are illustrated in Fig. 4. In Fig. 4(A), the signals from the haem peripheral protons of Dolabella metMb are resolved at > 39.9 ppm and were assigned previou~ly.~' The resonances of Dolabella metMbN, are observed at <38 ppm. The saturation of the 8-Me signal of metMb exhibits a large ST (ST factor lst/leq x 0) to a methyl proton signal at 21.6 ppm and small STs to the

E

Figure 3. Downfield hyperfine shifted portions of the 270 MHz 'H MNR spectrum and ST difference spectra of the mixture of equine metMb and met MbN; at pD 8.23 and 35°C. (A) Refer- ence spectrum. The signal assignments of the haem methyl proton resonances26 are indicated with the spectrum. The arrow indicates the peak being saturated and the signal due to the off-resonance saturation is indicated by an asterisk. (B) ST difference spectrum on saturation of the 8-Me signal of metMb. The ST connectivity to a haem methyl proton signal a t 25.1 ppm is observed. (C) ST dif- ference spectrum on saturation of the 5-Me signal of metMb. The ST connectivity to a methyl proton signal at 31.9 ppm is observed. (D) ST difference spectrum on saturation of the 3-Me signal of metMb. The ST connectivity to a haem metyl proton signal at 10.8 ppm is observed. (E) ST difference spectrum on saturation of the I -Me signal of rnetMb. The ST connectivity to a haem methyl proton signal at 25.8 pprn is observed. The ST connectivities in (B)-(E) clearly indicate the haem methyl proton signal assign- ments for equine metMbN,- as shown in (A).

resonances at 24.0 and 31.8 ppm, indicated by circles. The large ST connectivity identifies the 8-Me signal of metMbN,- and the small ST on the resonance at 31.8 ppm arises from the off-resonance saturation of the 5-Me signal of metMb, indicated by an asterisk in Fig. 4(B) (see below). A signal from the reversely oriented haem5 of metMb resonates under the 8-Me signal of metMbZ4 and therefore the ST on the resonance at 24.0 ppm is likely to arise from the corresponding resonance in metMbN,-. The saturation of the 5-Me signal of metMb exhibits a large ST to a haem methyl proton resonance at 31.8 ppm, clearly identifying the 5-Me signal of metMbN,- [Fig. 4(C)]. There are two small STs to the 8-Me signal and a resonance at 35.4 ppm of metMbN,-. The former arises from the off-resonance saturation of the 8-Me signal of metMb and, since a signal from the reversely oriented haem5 of metMb also resonates under the 5-Me signal of metMb, the latter is likely to arise from the connectivity between the signals of the reversely oriented haem in both forms of Mb. The ST connectivities in Fig. 4(D) and (E) clearly demon- strate the assignments of the 3- and 5-Me signals in met MbN, - .

PARAMAGNETIC 'H NMR SATURATION TRANSFER STUDY

metMb i iii 1

s11

i ~ " " ' ' ' ' I ' " ' I ' ' ~ ~ I ' ' 100 80 M) 40 a

B

Figure 4. Downfield hyperfine shifted portions of the 270 MHz 'H NMR spectrum and ST difference spectra of the mixture of Dolabella metMb and met MbN; at pD 7.24 and 30°C. [metMbN;]/[metMb] = 1.2. (A) Reference spectrum. The signal assignments of the haem methyl proton resonancesz7 are indicated with the spectrum. The arrow indicates the peak being saturated and the signal due to the off-resonance saturation is indicated by an asterisk. (6) ST difference spectrum on saturation of the 8-Me signal of metMb. The large ST connectivity to a haem methyl proton signal at 21.6 ppm and small STs to a haem methyl proton resonance at 31.8 ppm and a resonance at 24.0 ppm, indicated by circles, are observed. The large ST connectivity identifies the 8-Me signal of metMbN;. The small ST to the resonance at 31.8 ppm is due to the off-resonance saturation effect to the 5-Me signal of metMb and the other small ST arises from the ST connectivity between the resonances of the reversely oriented haem in both forms (a signal from the reversely oriented haem5 in metMb reson- ates under the 8-Me signal of metMb).24 (C) ST difference spec- trum on saturation of the 5-Me signal of metMb. A large ST connectivity provides the assignment of a methyl proton signal at 31.8 ppm to the 5-Me signal of metMbN;. Small STs to the 8-Me signal of metMbN; and a resonance at 35.4 ppm, indicated by circles, are due to the off-resonance saturation of the 8-Me signal of metMb and the connectivity between the resonances of the reversely oriented haem in both forms (a signal from the reversely oriented haem5 in metMb resonates under the 5-Me signal of rnetMb).Z4 (D) ST difference spectrum on saturation of the 3-Me signal of metMb. The ST connectivity to a haem methyl proton signal at 12.2 ppm is observed. (E) ST difference spectrum on saturation of the 1 -Me signal of metMb. The ST connectivity to a haem methyl proton signal at 23.9 ppm is observed. The ST factor observed in (8)-(E) is almost zero. These ST connectivities indi- cate the haem methyl proton signal assignments in Daoabella metMbN; as shown in (A).

The results of the ST experiments with saturation of the haem methyl proton resonances of Dolabella metMbN,- are given in Fig. 5. The saturation of the haem methyl proton resonances of metMbN,- yields the ST connectivities to the corresponding signals of metMb. However, the ST factor of 0.45 is much larger than the values observed for Fig. 4(B)-(E). Since z is almost equivalent for both forms under these experi- mental conditions, the difference in the ST factor

Figure 5. Downfield hyperfine shifted portions of the 270 MHz 'H NMR spectrum and ST difference spectra of the mixture of Dolabella metMb and met MbN; at pD 7.24 and 30°C. [metMbN;]/[metMb] = 1.2. (A) Reference spectrum. The arrow indicates the peak being saturated and the signal due to the off- resonance saturation is indicated by an asterisk. The + sign shows the ST connectivity from resonances other than that indicated with an arrow. (B) ST difference spectrum on saturation of the 5-Me signal of metMbN;. (C) ST difference spectrum on saturation of the 1-Me signal of metMbN;. (D) ST difference spectrum on saturation of the 8-Me signal of metMbN;. (E) ST difference spectrum on saturation of the 3-Me signal of metMbN,-. On the saturation of the haem methyl proton resonances of metMbN;, the STs, with an ST factor of about 0.45, are observed on the cor- responding haem methyl proton signals of metMb.

between Figs 4 and 5 is attributed to the difference in TYIr between metMb and metMbN,-.

The problem of the decoupler power spillage can be avoided in a 2D exchange experiment. A portion of the 2D exchange spectrum of the mixture of Dolabella metMb and metMbN,- at 30°C is given in Fig. 6. In the spectrum, the cross peaks a-d indicate the connecti- vities between the corresponding haem methyl proton resonance of metMb and metMbN, -. Additionally, the cross peaks e-j connect the resonances of the other resolved haem peripheral proton resonances. With the known assignments in Dotabella metMb,27 the corre- sponding resonances in metMbN, can be straightfor- wardly and unambiguously assigned from these connectivities. The signal assignments obtained are summarized in Table 1.

Effect of N 3 - exchange on p:tr of the 5-Me signal in Dolabella metMb and metMbN,-

The partially relaxed spectra resulted from the satura- tion of the 5-Me signal of Dolabella metMb at pD 7.00 and 30°C are shown in Fig. 7. The signal intensity recovers exponentially with time after the saturation

s12 Y YAMAMOTO Y. INOUE AND T. SUZUKI

i I 7-

Figure 6. Portion of the ' H 2D exchange spectrum of the mixture of Dolabella metMb and metMbN; ([metMbN,']/[metMb] = 1.2) at pD 7.24 and 30°C. A mixing time of 5 ms was used. The signal assignments in Dolabella metMb2' are indicated with the 1D spectrum. The cross peaks a-d indicate the connectivities between the haem methyl proton resonances of the two forms and the other haem peripheral proton resonances are connected by the cross peaks e-j. a, 8-Me; b, 5-Me; c, 3-Me; d, 1-Me; e, 7-a; f, 6-a; g, 4-1; h. 6-a'; i, 7-a'; j, 2-a.

and the TYtr value of 8.3 & 1.0 ms was calculated using the standard equation. 'The Ti;,' values of the 5-Me signal Dolabella metMbN, and the mixture of the two forms were similarly determined and are given in Table 2.

The apparent T p values of both Dolabella metMb and metMbN,- decrease in the presence of the ligand exchange, because the transfer of magnetization between the two forms of Mb via the ligand exchange accelerates the signal recovery. The substitution of TFtr (8.3 f 1.0 ms) for the 5-Me signal of Dolabella metMb and the ST value of 0.45 into Eqn (1) yielded a T-' (= k,,,) value of 150 f 50 s - ' . Then the k,, value of (1.4 & 0.5) x 106 1 mol- ' s - ' was calculated from kerf

5-Methyl h

. 2u 000

ms ppm

-7 ' I ' ' ' 7 - T - 7 100 90 80

Figure 7. Partially relaxed spectra of the 5-Me ' H N M R signal in Dolabella metMb at pD 7 00 and 30°C after selective saturation The signal intensity recovers exponentially with time and a TI"" value of 8.3 * 1 .O ms was calculated

[metMbN,-]/[metMb] (= 1.22) and LN3-J (=0.13 mM). Careful scrutiny on the kinetics of the ligand exchange using the ST experiments will be published elsewhere.

Signal assignments in Mustelus metMbN, connectivities

via ST

The ST difference spectra of the mixture of Mustelus metMb and metMbN, ~ ([metMbN,-]/[nietMb] =

Table 2. T'Ftr values (ms) for the 5-Me resonances of Dolu- bella metMb and metMbN,- at 30°C

merMb Mixture (pD 7 24) metM bN (pD 7 0) ([merMbN,-]/[metMb] - 1 2) (pi3 7 50)

8 3 i 1 . 0 4.7 * 1 2 - metMb metM bN, - - 1 3 * 1 5 21 1 1 5

Table 1. Assignments and chemical shifts (ppm) of the Mbs used

Dolabeib Mb(pD 7 24. 30 C) Musfelus Mb(p0 7 56 30°C) Equine Mb(p0 755 35°C)

Assignment metMb' meiMbN,' metMba metMbN; merMb merMb(lm)

1 -Me 55.4 3-Me 76.0 5-Me 88.7 8-Me 96.6 2-a 39.9 4-a 51.5 6-a 53.8 6-a' 48.0 7-a 61.3 7-a' 45.1

a Ref . 27,

'Assigned via NOE. Tentative assignment

23.9 12.2 31.8 21.6 9.9

19.6 13.0 14.9 7.3 5.5

54.6 74.0 87.4 97.9 34.5 45.2 59.9 39.3 81.3 40.5

27.7 50.1 26.5 16.5 69.1 7.4 34.9 81.2 39.0 28.6 87.9 17.9 1 9.3b

24.5' 12.7" 14.0" 13.1

PAKAM4GhL'i i i ' ' H NMK 5 4 1 URAlION T R A h S t t R STUDY S i 3

Figure 8. Downfield hyperfine shifted portions of the 2'70 MHz 'H NMR spectrum and ST difference spectra of The mixture of Mustelus metMb and metMbN; ([metMbN,-]/[nietMb] = 0.52) at pD 7.56 and 30°C. (A) Reference spectrum. The haem methyl proton signal assignments of Mustelus nietMb was reported pre- v i o ~ s l y . ~ ~ Peaks indicated by are due to impurity. The arrow indicates the peak being saturated and the signal due to the off- resonance saturation is indicated by an asterisk. (6) ST difference spectrum on saturation of the 8-Me signal of metMb. The ST con- nectivity is observed on a haem methyl proton signal at 28.6 ppm. (C) ST difference spectrum on saturation of the 5-Me signal of metMb. The ST connectivity is observed on a haem methyl proton signal at 34.9 ppm. (D) ST difference spectrum on saturation of the 3-Me signal of metMb. The ST connectivity is observed on a haem methyl proton signal at 16.5 ppm. (E) ST difference spec- trum on saturation of the 1 -Me signal of metMb. The ST connec- tivity is observed on a haem methyl proton resonance at 27.7 ppm. These ST connectivities clearly identify the haem methyl proton signal assignments in Mustelus metMbN; as shown in (A).

0.52) at pD 7.56 and 30°C are illustrated in Fig. 8. The signal assignments in Mustelus metMb has been report- ed p r e v i o u ~ l y . ~ ~ In Fig. 8(B)-(E), the saturation of the haem methyl proton signals of metMb exhibits the ST to the corresponding haem methyl proton resonances of metMbN, - _ Hence the haern niethyl proton resonances of metMbN, ~ are assigned from these connectivities. The ST connectivities for the haem methyl proton res- onances can be also observed in the 2D exchange spec- trum shown in Fig. 9. The connectivities for the other haem peripheral proton resonances cannot be identified in the present 2D map. However, the haem peripheral proton resonances in metMbN,- could be assigned via NOE (results not shown), based on the haem methyl signal assignments obtained. The assignments in Mus- telus metMbN,- are given in Table I.

Signal assignments in equine metMb(1nr) via ST connectivities

a.

Figure 9. Portion of the 'H 2D exchange spectrum of the mixture of Mustelus metMb and metMbN,- ([metMbN,-]/[metMb] = 0.52) at pD 7.56 and 30°C. A mixing time of 10 ms was used. The signal assignments in Mustelus metMbZ7 are indicated with the 1 D spectrum. The cross peaks a-d indicate the connectivities between the haem methyl proton resonances of the two protein forms. a, 8-Me; b, 5-Me; c. 3-Me; e, 1 -Me.

Figure 10. Downfield hyperfine shifted portions of the 270 MHz 'H NMR spectrum and ST difference spectra of the mixture of equine metMb and metMb(lm) ([metMb(lm)]/[metMb] = 6.4) a t pD 7.55 and 35°C. (A) Reference spectrum. The arrow indicates the peak being saturated and the signal due to the off-resonance saturation is indicated by an asterisk. (6) ST difference spectrum on saturation of the 8-Me sianal of metMb. (C) ST difference ~ .~ ~~ ~

spectrum on saturation of the ;-Me signal of metMb. (D) ST dif-

equine metMb and Mb(1m) ([metMb(Im)]/ ST connectivities in (6)-(D) identify the 8-, 5- and 1 -Me signals [metMb] = 6.4) at pD 7.55 and 35 "C are illustrated in

The ST connectivities observed for the mixture Of ference spectrum on Saturation of the 1 -Me signal of metMb, The

of metMb(lm), respectively. The ST factor is about 0.93.

S14 Y. YAMAMOTO Y. INOUE AND T. SUZUKI

metMb(lm)

60 60 40 20

a, b

D B

C B

I ’ d

0 Q

B

Figure 11. Portion of the ’H 2D exchange spectrum of the mixture of equine metMb and metMb(1m) ([metMb(lm)]/ [metMb] = 1 .I ) at pD 7.47 and 35°C. A mixing time of 5 rns was used. The cross peaks a 4 indicate the connectivities between the haem methyl proton resonances of the two protein forms. a, 8-Me; b, 5-Me; c, 3-Me; d, 1 -Me.

Fig. 10. The signals from the haem peripheral protons in metMb(1m) are resolved at <40 ppm. The saturation of the 8-Me signal of metMb exhibits the ST connec- tivity, with an ST factor of 0.93 to a haem methyl proton signal of metMb(1m) at 17.9 ppm. Figure 1O(C) and (D) similarly identify the 5- and 1-Me signals of metMb(Im), respectively. The 3-Me signal of metMb(1m) could not be located in the ST difference spectrum owing to a bad cancellation of the signals in the diamagnetic envelope. In the 2D exchange spectrum shown in Fig. 11, the cross peak c clearly identifies the location of the 3-Me signal in metMb(1m). The assign- ments of the haem methyl proton signals in equine metMb(1m) are given in Table 1.

DISCUSSION

exceptions for OH l i g a t i ~ n , ~ ’ . ~ ~ if the paramagnetic relaxation rate is smaller than the ligand exchange rate.

The hyperfine shift of the haem methyl proton reson- ance in metMb is composed of contact and pseudo- contact shifts. Although the essentially axial pseudo-contact shift arising from the substantial zero- field splitting characteristic of the ‘ A state of iron(II1) haem proteins3‘ could contribute significantly to the hyperfine shift, it cannot be responsible for the spread of the haem methyl proton resonances. Therefore, the hyperfine shift pattern of the haem methyl proton res- onances in metMb is interpreted in terms of the contact shift and hence reflects the in-plane asymmetry of the haem electronic structure. The distribution of unpaired electrons among the four pyrroles is directly perturbed by the axial ligand orbital. However, with the limited variation in the angle between the projection of the proximal histidylimidazole plane on to the haem plane and the N-Fe-N axis, among the myoglobins so far s t ~ d i e d , ~ ’ , ~ ~ the hyperfine shift pattern of the haem methyl proton resonances in these metMbs was found to be independent of the p r ~ t e i n . ~ ~ . ~ ’ Consequently, the ST connectivities directly provide the specific haem methyl proton assignments of low-spin form of Mb, which in turn allow the determination of rhombic anisotropy of the haem electronic structure. The ST connectivities are successfully utilized in connecting the hyperfine shifted haem peripheral proton resonances of metMb and metMbN3- or metMb(1m). However, no ST connectivity is observed for CN- ligation (results not shown), owing to the violation of the second condi-

is about 80 ms for a haem methyl proton resonance of met-cyano Mb” and 7 - l (=k,,,) for CN- ligation is < 1 s - ’ , ~ ’ indicating that ST experiments cannot be considered as a general method for signal assignments in paramagnetic Mb.

The ST connectivities can be observed in both 1D and 2D methods. Obviously, the 1D ST difference spec- trum has the advantage in sensitivity. The problem of the decoupler power spillage can be overcome in a 2D exchange spectrum. However, competition, during the mixing time, between the build-up of the ST connec- tivity and the decay of magnetization due to paramag- netic relaxation may result in a poor signal-to-noise ratio of the 2D spectrum.

tion; T i n t r .

Kinetics of N,- binding to Dolabella metMb

Signal assignments of haem peripheral proton resonances in paramagnetic Mb by ST connectivities

Since, in addition to the constraint z < TYr, indepen- dent observation of the signals arising from each species is essential for ST experiments, the difference (Ad) in the resonance frequencies between the two forms of Mb must be much larger than the ligand-exchange rate, i.e., 7-1 4 Ah. Hyperfine shifted resonances are associated with a large chemical shift range and therefore ST experiments are advantageously used in connecting iron(II1) high- and low-spin forms of Mb, with some

Dolabella metMb possesses a Val residue at the distal E7 site, instead of the usual His residue.29 A recent study has revealed that the guanidino NH proton of the Arg El0 in this M b is hydrogen bonded to the Fe- bound ligand.” The Arg El0 in Dolabella Mb, acting as the ‘pseudo-distal’ residue, appears to be responsible for the control of the ligand affinity in this Mb. The molec- ular mechanism for ligand stabilization in Dolabella Mb is very similar to that found in Mb from the sea hare Aplysia limacina, which also possesses the distal Val E7 residue.40 Therefore, the functional properties of the mollusc Mb are controlled by a mechanism completely different from that of the usual Mb with the distal His E7 residue.

PARAMAGNETIC 'H NMR SATURATION TRANSFER STUDY S15

The k,, and koff values of (1.5 0.4) x lo6 1 mol-' s-' and 150 50 s-', respectively, for Dolabella metMb at pD 7.24 and 30°C are close to the values reported for Aplysia metMb (ken = 1.8 x lo6 1 mol-' s-', koff = 750 s C 1 at pH 6.15 and 20°C)41 and are dif- ferent from those for equine metMb (k,, = 2.6 x lo3 1 mol-' s - l , koff = 0.38 s-' at pH 7.0 and 20"C).35 An x-ray study revealed that the coordination geometry of N,- to the haem iron and the hydrogen bonding inter- action between the Fe-bound N,- and the distal amino acid residue in Aplysia metMbN,- are different from those found in mammalian metMbN,-.42 The larger k,, and smaller koff values for Aplysia metMbN,- than the corresponding values of equine metMbN, - have been explained by the 'open' structure in the distal site of ligand-free Aplysia metMb and the steric repulsion between the Arg El0 and the Fe-bound N3-, respec- t i ~ e l y . ~ ~ , ~ ~ With a reasonable assumption of the struc- tural similarity between Aplysia and Dolabella Mbs,' 5 7 2 0 * 2 4 the kinetic data for Dolabella metMbN, may be similarly interpreted.

Hyperfine shift pattern of haem methyl proton resonances in metMbN,-

Owing to the intermediate field strength of the N,- ligand, metMbN,- exhibits the thermal spin equi- librium between the two different spin states, i.e. S = 5/2 and 1/2.32*44-46 S ince the NMR spectra of equine metMbN, - have been analysed only Dolabella and Mustelus metMbN, s are considered here. The thermal spin equilibrium in metMbN3- can be characterized from the analysis of the haem methyl proton resonance^.^'-^^ The hyperfine shift patterns of the haem methyl proton resonances of Dolabella and

Mustelus metMbN3- s are 5-, 1-, 8-, 3-Me and 5-, 8-, 1-, 3-Me in order of decreasing chemical shifts, respectively (see Table 1). Since the shift pattern for purely high-spin meth4b is 8-, 5-, 3-, l-Me,26927 the difference in the shift order of 1- and 8-Me between the two nietMbN,- s is attributed to a greater fraction of high-spin state in Mustelus metMbN, -, which is also supported by its greater average (26.9 ppm) and smaller spread (18.4 ppm) of the haem methyl proton hyperfine shifts. The greater high-spin fraction in Mustelus metMbN, - than in Dolabella metMbN,- may stem from the weaker bonding interaction between the haem and the axial ligand. A detailed analysis of the temperature depen- dence of the haem methyl proton resonances will be published elsewhere.

CONCLUSIONS

The 1D and 2D ST experiments can be used in connect- ing iron(II1) high- and low-spin forms of Mb via ligand- exchange reactions. The constraints for the detection of the ST connectivity restricts the applicability of this methodology to the system of ligands with intermediate affinity to the haem iron. Quantitative analysis of the ST factor leads to the characterization of the kinetics of the ligand exchange.

Acknowledgement

We thank Professor Martino Bolognesi (University of Pavia) for making Ref. 44 available to u s prior to publication.

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