Eur. J. Biochem. 78, 133- 140 (1977)
`
`Structural Rearrangements Due to Ligand Binding and Haem
`Replacement in Myoglobin and Leghaemoglobins
`
`Nico? A. NICOLA and Sydney J. LEACH
`Department of Biochemistry, University of Melbourne
`
`(Received July 30, 1976/June 1, 1977)
`
`Structural rearrangements in sperm whale myoglobin and leghaemoglobins caused by changes in
`the spin or oxidation state of the iron as a consequence of ligand binding have been measured by
`difference spectroscopy in the ultraviolet. When compared with the high-spin acetate complex,
`ligands which cause a transition to the low-spin state also cause large perturbations of tyrosine(s)
`remote from the haem pocket in myoglobin but only minor perturbations of tryptophan(s) in legha-
`emoglobin. This may indicate a weaker coupling between events at the haem site and conformational
`changes in the protein in leghaemoglobins. The absorption spectra of various haem-liganded forms
`of the two proteins as well as the binding of the dye rose Bengal to the two apoproteins are consistent
`with weaker interactions between the haem and apoprotein and a more solvent-exposed haem pocket
`in leghaemoglobin compared with myoglobin.
`
`Leghaemoglobins are plant haemoproteins which
`resemble animal myoglobins with respect to their
`molecular weight, monomeric state and their ligand-
`binding (especially oxygen-binding) properties. Their
`high oxygen affinity is presumed to be central to their
`biological function in the root nodules of legumes (see
`e.g. [I]). Most of the reported structural, spectroscopic
`and ligand-binding studies have been on the major
`leghaemoglobin component from soybean (Glycine
`max, cr Lincoln). It has recently been shown that this
`plant produces several leghaemoglobins of differing
`amino acid sequence but similar overall conformation
`[l, 21, that these characteristics are probably general
`in Leguminosae (Thulborn et al., unpublished) and that
`the most readily discernable differences between
`leghaemoglobins of widely different origin and amino
`acid sequence, is their antigenicity [3]. Comparing
`the leghaemoglobins with sperm whale myoglobin
`reveals interesting similarities and differences [2,4].
`For the purposes of the present paper, the most
`pertinent conclusion was that the haem moiety may
`be more loosely held in the leghaemoglobins than in
`myoglobin and there is less resistance to changes in
`spin state on ligand binding in the former case. This
`is consistent with the observations that soybean
`leghaemoglobin binds nicotinic acid with much higher
`affinity [5,6] and its haem moiety is more readily
`exchangeable [7] than in myoglobin. Evidence has
`
`Abbreuiution. CD, circular dichroism.
`
`also been adduced that the haem pocket in the plant
`globin is not as non-polar as that in myoglobin [8,9].
`The purpose of the studies described here was to
`seek evidence of structural changes occurring in both
`types of haem proteins arising from changes in spin
`state. The reason for our interest in such changes is
`the observation by Perutz [lo] that high-spin to low-
`spin transitions in tetrameric haemoglobin are ac-
`companied by deep-seated molecular rearrangements
`but little is known of corresponding changes in
`monomeric systems. In order to compare them with
`myoglobin and to generalise from the results, several
`leghaemoglobins and a variety of ligands (including
`nicotinate) and both oxidation states were used.
`Changes in absorption spectra in the visible, Soret and
`near-ultraviolet were measured, usually by the dif-
`ference technique. Finally, the dye rose Bengal was
`used as a spectroscopic indicator of the polarity of the
`haem pockets in the two types of proteins.
`
`MATERIALS AND METHODS
`Materials
`The dye rose Bengal(3’4’5’6’-tetrachloro-2,4,5,7-
`tetraiodofluorescein disodium salt) was obtained from
`Fluka. Chlorohemin was lot 8071 from Nutritional
`Biochemicals Corp. and was recrystallised according
`to Fischer [13 1. Leghaemoglobins were extracted and
`purified as already described [2,4]. Sperm whale
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1044, Page 1 of 8
`
`

`

`134
`
`myoglobin was batch 10 from Seravac or batch 52348
`from Koch-Light.
`Potassium ferricyanide, potassium cyanide, potas-
`sium cyanate, sodium acetate, sodium azide, sodium
`dithionite and sodium fluoride were AR grade from
`British Drug Houses. Nicotinic acid was AR grade
`from Townson and Mercer and imidazole from Sigma.
`Other chemicals were AR grade and glass-distilled
`water was used throughout.
`
`Concentration Estimations
`Absorption spectra for the reduced pyridine
`haemochromes of soybean, serradella and lupin leg-
`haemoglobins were identical with those for sperm
`whale myoglobin. Since the spectra for other com-
`plexes of these proteins were much more variable than
`this, the formation of pyridine haemochromes was
`used as a measure of haemoprotein concentration as
`in previous studies [2,4].
`
`Formation of Liganded Components
`Most liganded complexes of leghaemoglobin and
`myoglobin were formed simply by titration with the
`appropriate ligand until no further spectral changes
`could be observed. The deoxyferrous and oxyferrous
`complexes were formed as described previously [4]
`and the percentage of oxyferrous and ferric forms was
`assayed as described in the same paper.
`
`Formation of Apoproteins
`Apoproteins of myoglobin and leghaemoglobins
`were made from the haem proteins by the acetone/HCl
`method of Rossi-Fanelli et al. [12]. Acetone (BDH)
`was redistilled from potassium permanganate and
`Drierite. Glass-distilled water was de-ionised using a
`Bio-Rad mixed-bed ion-exchange resin. If the removal
`was carried out at -20 "C the products usually had
`less than 1% of the original haem content as judged
`from the A,oo/A,8, ratio and were recovered in about
`70% yield. Their concentrations and those of the haem
`proteins were determined as previously described
`[2,4]. Molar absorption coefficients at 280 nm in
`phosphate pH 7.5 were 17900 and 16300 M-' cm-'
`for soybean apoleghaemoglobin and sperm whale
`apomyoglobin respectively.
`
`Absorption Spectra and Difference Spectra
`Absorption spectra were measured with a Cary 14
`spectrophotometer. Difference spectra were usually
`run on this instrument using conditions similar to
`those reported by Nicola and Leach [13] except that
`a four-cell arrangement was usually used to allow
`
`Structural Effects of Liganding in Myoglobin and Leghaemoglobins
`
`compensation for the addition of absorbing ligands
`to the sample cell.
`
`Circular Dichroic Spectra
`The instruments and procedures used for measur-
`ing CD spectra were those described by Nicola et al.
`141.
`
`RESULTS
`Effect of Spin and Oxidation State on Soret and
`Visible Absorption Spectra
`Spectral data for soybean leghaemoglobin a and
`the main lupin component have been reported by a
`number of workers [1,9,14- 161 and these have been
`supplemented by data for the aquoferric, fluoroferric
`and nicotinoferric as well as the oxyferrous and deoxy-
`ferrous soybean derivatives [4]. In the present work,
`Soret and visible absorption spectra have been
`measured for the acetate, cyanate, azide and imidazole
`derivatives of ferric soybean leghaemoglobin. All of
`these spectra are best discussed by grouping the deri-
`vatives as follows: high-spin ferrous = deoxy ; low-
`spin ferrous = oxy, pyridine and nicotinate; high-spin
`ferric = acetate and fluoride; low-spin ferric = cyanide,
`imidazole and nicotinate ; mixed-spin ferric = aquo,
`cyanate, azide.
`The spectra for soybean leghaemoglobin a are very
`similar to those reported for lupin leghaemoglobin [9].
`They are also quite similar in general form to those
`for sperm whale myoglobin [17] with corresponding
`complexes showing the same spin-state tendencies.
`However, there are differences in the peak absorption
`intensities and their wavelengths. Table 1 shows that
`the Soret band for all classes of leghaemoglobin
`spectra consistently occurs at shorter wavelengths
`(blue shift) than for corresponding complexes of
`myoglobin independent of the spin or oxidation state
`of the iron. Also the mixed-spin complexes for leg-
`haemoglobin appear to be further towards the low-
`spin state than those for myoglobin (not shown).
`
`Binding of Nicotinic Acid
`The binding of nicotinic acid with relatively high
`affinity to both ferrous and ferric leghaemoglobin is
`of some interest because this ligand does not bind to
`myoglobin except at much higher concentrations [18]
`and because it has been postulated that it may play a
`regulatory role in viuo. This hypothesis and the binding
`of nicotinic acid to soybean leghaemoglobin a has
`been discussed in detail by Appleby et al. [1,5,6]. We
`have extended these observations to show that the
`high-affinity binding of nicotinic acid is probably a
`general phenomenon for leghaemoglobins. Thus, leg-
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1044, Page 2 of 8
`
`

`

`Nicos A. Nicola and Sydney J. Leach
`
`haemoglobins from serradella, snake bean and lupin
`all showed changes in absorption spectra for their
`ferrous forms (reduced with sodium dithionite) when
`titrated with nicotinic acid in 0.01 M phosphate buffer,
`pH 7.0, which were qualitatively similar to those of
`the soybean protein [5,6]. The structural consequences
`of nicotinic acid binding are best seen using difference
`spectra. Fig. 1 shows such a set of spectra for the
`titration of ferric soybean leghaemoglobin a in the
`near-ultraviolet (Fig. 1 A) and visible (Fig. 1 B) regions.
`The visible difference spectra show the progressive
`decrease in the charge-transfer band intensities at 494
`band
`and 629 nm and the increase in the ct and
`intensities at 530 and 556 nm respectively. This is
`typical for a high-spin to low-spin transition.
`The changes which occur concomitantly in the
`near-ultraviolet, (Fig. 1 A), are perhaps more interest-
`ing. These changes run parallel with those in the visible
`
`Table 1. Wavelengths of the main Sorzt peak for various complexes
`sperm whale myoglobin, soybean leghaemoglobin a and lupin
`of
`leghaemoglobin a
`
`Complex type
`
`Ligand Wavelength for
`
`sperm whale soybean
`Lb
`Mb [I71
`
`lupin
`Lb PI
`
`nm
`
`423
`406
`410
`418
`434
`
`41 7
`403
`403
`412
`427
`
`416
`403
`404
`-
`42 1
`
`Low-spin ferric
`High-spin ferric
`Mixed-spin ferric
`Low-spin ferrous
`High-spin ferrous
`
`CN
`F
`H,O
`0 2
`-
`
`135
`
`and, in fact, some of this difference absorption is due
`to spectral changes in the haem. Strickland et al. [I91
`however, have shown that neither the haem nor the
`haem undecapeptide of cytochrome c (which contains
`no aromatic amino acids) display any fine structure
`bands in the haem absorption region between 250 and
`310 nm, even at liquid nitrogen temperatures, SO the
`haem cannot be the origin of the sharp difference
`peaks seen in Fig. 1 (A). The peaks below 280 nm may
`well arise from the transfer of nicotinic acid from an
`aqueous environment into the non-polar haem pocket,
`since the parent spectrum of nicotinic acid shows
`peaks at 270, 264 and 257 nm. However, the negative
`difference peak at 294 nm and probably the one at
`286 nm must arise from a change in the environment
`of a tryptophan residue and this change is proportional
`to the extent of binding of nicotinate. Such negative
`difference peaks, superimposed on an increasingly
`positive haem background, suggests that the trypto-
`phan(s) become more exposed to aqueous solvent as
`the titration proceeds.
`
`‘Form’ DifSerence Spectra in the Near- Ultraviolet
`‘Form’ difference spectra ( i e . the difference spectra
`for various liganded complexes against the pure high-
`spin acetate complex) were recorded in the near-ultra-
`violet for both sperm whale myoglobin and soybean
`leghaemoglobin a (see Fig. 2). As mentioned before,
`haem itself shows no fine structure bands between 250
`and 310 nm as evidenced by the difference spectrum
`for low-spin ferric haem cyanide versus high-spin
`ferric haem chloride. On the other hand, difference
`
`0 06
`
`A
`
`556
`
`0 12
`
`0 10
`
`0 00
`
`0 06
`
`0 04
`
`T
`4
`0 02
`
`0
`
`-0 0 2
`
`-0 04
`
`-0 06
`
`Wavelength (nm)
`Fig. 1. Difference spectral titration of soybean ferric leghaemoglobin a with nicotinic acid [ A ) in the near-ultraviolet and (B) in the visible.
`Leghaemoglobin concentration was 33.4 BM in 0.01 M potassium phosphate buffer, pH 5.4, and each addition (1,2,3,4) increased the con-
`centration of nictonic acid by 11.6 pM. Difference spectra were performed with a four-cell arrangement that allowed blanking of both protein
`and ligand. Saturation was achieved at a final concentration of 58 pM nicotinic acid (not shown)
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1044, Page 3 of 8
`
`

`

`136
`
`A q
`
`Structural Effects of Liganding in Myoglobin and Leghaemoglobins
`
`2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 31
`2 4 0 260 2 8 0 3 0 0 320 3 4 0 31
`Wavelength (nm)
`Wavelength (nm)
`Fig. 2. Form difference spectra for ( A ) sperm whale ferric myoglobin and ( B ) soybean ferric leghaemoglobin a liganded complexes. For (A)
`A A of 0.05 corresponds to a As of 1.9 mM-' cm-'; for (B) A A of 0.05 corresponds to a Ae of 2.3 mM-' cm-'. All complexes were
`measured against the corresponding acetate complex as reference. The difference spectra are labelled according to the bound ligand. Some
`of these difference spectra have been offset vertically for clarity and the baseline position for each marked with a dash through the curve
`on the long-wavelength side. The dashed curves between 270 and 295 nm are inferred background absorptions due to the haem. For reference,
`the curve (0-0) for cyanoferric haem (haemin cyanide) versus haemin chloride is also shown. All samples were in 0.1 M sodium acetate
`buffer, pH 5.2, and liganded complexes were formed by titration with solid ligand until no further spectral changes were observed. Leghaemo-
`globin a concentration was 22 pM and myoglobin concentration 26 pM at 20 "C. A four-cell arrangement was used
`
`3
`
`spectra of both myoglobin and leghaemoglobin low-
`spin ferric complexes versus the high-spin acetate
`complex do show fine structure difference bands in
`this spectral region and these must reflect changes in
`the environments of the aromatic amino acids upon
`such a transition. However, there is a difference be-
`tween the responses of myoglobin and leghaemoglobin
`to a change in the sixth ligand.
`On formation of a low-spin complex from a high-
`spin one in myoglobin there is a large difference ab-
`sorption attributable to aromatic amino acids (see
`especially the cyanide complex versus the acetate) with
`peaks at 280 and 286 nm. These are most likely to arise
`from the exposure of a buried tyrosine to aqueous
`solvent since tryptophan would give rise to additional
`peaks at higher wavelengths.
`Replacement of one high-spin ligand for another
`in myoglobin (fluoride or cyanate for acetate) shows
`a much smaller aromatic contribution to the difference
`spectrum. The azide complex of myoglobin which is
`mixed-spin but predominantly low-spin appears not
`to follow the expected pattern if the proposed back-
`ground absorption that we have drawn is correct.
`The form difference spectra for leghaemoglobin
`(Fig. 2B) show much smaller aromatic contributions
`than do those for myoglobin. The wavelengths of the
`difference peaks are, however, indicative of a change in
`the environment of tryptophan rather than of tyrosine,
`
`as was observed for the nicotinate versus aquoferric
`complex in Fig. 1A. Since tryptophan difference
`spectra are expected to be three times larger than for
`tyrosine, given the same change in environment [13,20],
`this means that the change in the tryptophan(s) en-
`vironment in leghaemoglobins is minor compared to
`that for the tyrosine(s) in myoglobins. Cyanate
`(mixed-spin) and fluoride (high-spin) gave aromatic
`peaks nearly as intense as those given by cyanide (low-
`spin), imidazole (low-spin) and azide (mixed-spin).
`
`Binding of Rose Bengal to the Apoproteins of
`Myoglobin and Leghaemoglobin
`Coulson and Yonetani [21] have used the anionic,
`aromatic dye rose Bengal as a sensitizer in photo-
`oxidation studies on a number of apohemoproteins
`including apomyoglobin. They showed that this dye
`bound strongly at one site but weakly at a number of
`other sites in all of these apoproteins and that this
`binding could be impaired by addition of stoichio-
`metric amounts of haem. We have used rose Bengal
`as a probe of the haem pocket in apomyoglobin and
`apoleghaemoglobin. Its main usefulness for this pur-
`pose is that it does not present any of the complications
`with spin and oxidation states that make it difficult to
`interpret unambiguously many of the changes that
`occur in haemoproteins.
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1044, Page 4 of 8
`
`

`

`Nicos A. Nicola and Sydney J. Leach
`
`A
`
`Haern.CN
`
`Rose Benaal
`
`I
`
`0 2
`
`01
`
`137
`
`I
`
`T
`o a
`
`-0 1
`
`I::.:
`
`1
`600
`
`650
`
`Rose Bengal or haem-CN/Mb (mol/mol)
`Fig. 4. Plot of the binding data for rose Bengal and cyanoferric haem
`to sperm whale apomyoglobin followed by diference spectroscopy.
`Curve A is the size of the difference peak at 407 nm when the apo-
`protein is titrated with cyanoferric haem. Curve B is the size of the
`difference peak at 568 nm when the apoprotein is titrated with rose
`Bengal. Curve C is the size of the difference peak at 568 nm when
`the result of curve A is titrated with rose Bengal. Apomyoglobin
`concentration was 6.9 pM in 3 ml sodium bicarbonate (50 mg/l,
`pH 7.5) and consecutive 1, 3 or 6 p1 additions of stock rose Bengal
`(2.91 mM) or cyanoferric haem (2.69 mM) were made each time
`
`1
`350
`
`I
`400
`
`I
`450
`
`I
`500
`Wavelength
`
`I
`550
`(nm)
`
`0.3
`
`0.2
`
`7
`4
`
`0.1
`
`it can be clearly seen that as the cyano-haem binds to
`the protein there is a stoichiometric release of bound
`rose Bengal. The binding of rose Bengal to apoleg-
`haemoglobin a and the competition with haem is
`plottedin Fig. 3B. This shows that rose Bengal binds
`to only one high-affinity site in apoleghaemoglobin a
`and since it is stoichiometrically released from this
`site by haem this is probably the haem pocket (though
`we cannot rule out a strong anticooperative linkage
`between two different sites).
`Fig. 4 shows the results of a similar binding study of
`rose Bengal to apomyoglobin. In confirmation of the
`conclusions of Coulson and Yonetani [21] it can be
`seen that there is one high-affinity site for rose Bengal
`in apomyoglobin but there are, in addition, a number
`of weaker binding sites. This is shown by curve (C)
`which indicates that binding of the dye will still occur
`even when the pocket is occupied by the haem.
`It is clear from these data that, if the rose Bengal
`concentration is held below that of the apoprotein of
`either myoglobin or leghaemoglobin, the dye will be
`bound only at the haem site and the spectral properties
`of such a complex between 400 and 600 nm will reflect
`the haem environment. Such spectra are shown for
`apomyoglobin and apoleghaemoglobin in Fig. 5. The
`spectrum of rose Bengal is shifted by about 20 nm to
`higher wavelengths when incorporated into these
`apoproteins and this is consistent with the transfer
`from aqueous solution to a non-polar environment
`as evidenced by the spectral changes that occur when
`rose Bengal is transferred from water to ethanol.
`Thermal perturbation difference spectra properties of
`these complexes are described by Nicola and Leach [8].
`
`
`
`0
`5
`4
`3
`2
`1
`0
`(mol/mol)
`Rose Bengal or haem-CN/Mb
`Fig. 3. Difference spectra produced when soybean apoleghaemoglobin
`is tirratedwith cyanojerric haem. (A) The
`a saruruted wirli rose Ben&
`curve labelled 0 is the difference spectrum for leghaemoglobin .
`rose Bengal uersus free rose Bengal. Curves 1 - 6 are the difference
`spectra produced by additions of cyanoferric haem (concentration
`of haem increasing by 0.9 pM on each addition). Apoleghaemo-
`globin a was 3.5 pM in sodium bicarbonate (50 mg/l adjusted to
`pH 7.5). A four-cell arrangement was used. (B) Plot of the binding
`data in (A). Curve I is a plot of the size of the difference peak at
`568 nm when apoleghaemoglobin a is titrated with rose Bengal
`(representing binding of the dye). Curves I1 show that when the
`result of curve I is titrated with cyanoferric haem the difference peak
`at 568 nm decreases in size while the difference peak at 407 nm
`(representing haem binding) increases in proportion
`
`In Fig. 3A is shown a typical haem competition
`experiment after rose Bengal had been bound to
`soybean apoleghaemoglobin a. The cyan0 complex of
`free haem was used because this is known to be
`monomeric in solution. The difference absorption
`band due to the binding of cyano-haem is well se-
`parated from that due to the bound rose Bengal and
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1044, Page 5 of 8
`
`

`

`Structural Effects of Liganding in Myoglobin and Leghaemoglobins
`
`I:
`
`-
`-100 -5
`E
`7 J
`5
`m
`71
`
`-200 D + I
`
`0
`
`t
`
`-300
`600
`
`5 0 0
`
`550
`Wavelength (nrn)
`Fig. 6 . Circular dichroism of rose Bengal bound to ( A ) sperm whale
`apomyoglobin and ( B ) soybean apoleghaemoglobin a. All samples
`were in 0.1 M potassium phosphate buffer, pH 7. Rose Bengal con-
`centration was (A) 0.104 mM and (B) 0.134 mM. In each case the
`apoprotein was added in a ratio just greater than equimolar. A
`solution of 0.15 mM rose Bengal gave no CD bands on its own.
`Cell pathlength was 2 mm
`
`138
`
`554nrn
`
`I --
`
`60
`
`I -
`5
`40 -
`i
`E
`v
`w
`
`2 0
`
`0
`
`Wavelength (nrn)
`-) water, (-)
`Fig. 5. Absorption spectra of rose Bengal in (-
`ethanol, (. . . . .) bound to apomyoglobin and (- ' . -) bound to
`apoleghaemoglobin a. Peak absorption wavelengths are indicated.
`For all spectra, rose Bengal concentration was 20 pM. Apomyo-
`globin and apoleghaemoglobin concentrations were also 20 pM in
`0.1 M sodium phosphate buffer, pH 7.0
`
`~
`
`~
`
`CD Effects with Rose Bengal
`
`The binding of rose Bengal had no significant
`effect on the far-ultraviolet CD spectrum of apomyo-
`globin but increased [ O ] , at 220 nm of soybean apo-
`leghaemoglobin from -16300 to -19300 deg. cm2
`dmol-' at pH 7.05, suggesting that the dye stabilises
`apoleghaemoglobin in much the same way as does the
`haem group [4].
`Fig. 6 shows the CD spectra for rose Bengal bound
`to apomyoglobin and apoleghaemoglobin a. A solu-
`tion of rose Bengal free in solution gave no CD peaks
`but it showed strong negative CD bands when bound
`to apoleghaemoglobin and much weaker negative CD
`bands when bound to apomyoglobin. The wavelength
`positions of the CD peaks in these two complexes
`show the same trend as seen in corresponding absorp-
`tion spectra: that is, the peaks are red-shifted on
`binding. It is not known in which direction the tran-
`sition moments of rose Bengal point when the dye is
`bound in the haem pocket but these data are consistent
`with the postulate that the protein side chains in
`leghaemoglobin interact with transitions in both the
`x and y directions of the haem to give negative rota-
`tional strengths whereas in myoglobin there is a
`substantial cancellation of positive and negative
`strengths so that the CD spectrum can be slightly
`positive or negative depending on which component
`predominates [22].
`
`The discrepancy between the positions of the ab-
`sorption and circular dichroism maxima for the rose-
`Bengal . apomyoglobin complex may be due to the
`fact that not all of the dye is bound. The absorption
`spectra would then record an averaged result for
`bound and unbound rose Bengal but the circular
`dichroism spectra would record only the bound dye
`because the unbound form is optically inactive. Thus,
`if binding is incomplete, the distinction between the
`polarity of the leghaemoglobin and the non-polarity
`of the myoglobin pocket is accentuated because the
`true absorption maxima would be 578 nm for rose-
`Bengal . apomyoglobin (from circular dichroism) and
`563 nm for rose-Bengal
`apoleghaemoglobin (from
`circular dichroism and absorption). It would, how-
`ever, decrease the large difference in ellipticity between
`the rose-Bengal . apomyoglobin and the rose-Bengal .
`apoleghaemoglobin complexes.
`
`+
`
`DISCUSSION
`The blue shift of leghaemoglobin absorption
`spectra relative to corresponding myoglobin spectra,
`by analogy with solvent effects on other chromophores
`(see e.g. [13,20]), probably indicates a more polar
`(aqueous) environment of the haem group in leg-
`haemoglobin (see also [8]). This idea is also supported
`by the blue shift of absorption and CD spectra of the
`rose-Bengal . apoleghaemoglobin complex relative to
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`Motif Exhibit 1044, Page 6 of 8
`
`

`

`Nicos A. Nicola and Sydney J. Leach
`
`4
`
`those of rose-Bengal . apomyoglobin if our assump-
`tion that rose Bengal binds in the vacant haem pocket
`is correct.
`Ultraviolet-difference spectra caused by replacing
`the sixth ligand in both haemoproteins indicate that
`such replacements perturb aromatic amino acids. The
`degree of such perturbations is difficult to assess be-
`cause of the necessity to subtract out the changes in
`haem absorbance. Because haem itself shows no fine
`structure changes in its spectrum between 260 and
`300 nm when the sixth ligand is changed, our approach
`to this problem has been to simply draw a smooth
`connecting line between spectral points at 270 and
`300 nm as an approximation; this is the kind of
`difference spectrum obtained for haemin cyanide
`(low-spin) versus haemin chloride (high-spin) when
`protein perturbations are absent (Fig. 2).
`For myoglobin, the perturbation of aromatic re-
`sidues is related to the spin state in that the low-spin
`complexes (cyanide, imidazole, and azide) show larger
`changes in tyrosine environment relative to the high-
`spin acetate complex than do the other high-spin
`complexes (cyanate, fluoride). For cyanide and imida-
`zole complexes at least, the difference sp ctra are
`consistent with exposure of a tyrosine resi ue (blue
`shift) in going from the high-spin to the low-spin state.
`For tetrameric haemoglobin Perutz [lo] has shown
`that the high-spin to low-spin transition occurring
`upon oxygenation is accompanied by the expulsion
`of Tyr-H22 from a buried position to an exposed one.
`Sperm whale myoglobin also has Tyr-H22 in the same
`position and it seems to serve the same function as in
`haemoglobin; namely, it forms a hydrogen bond to a
`peptide carbonyl (FG-5), thus cross-linking the F and
`H helices [23], so that this change is probably the
`source of the observed difference spectra.
`In contrast to this, the difference spectra for leg-
`haemoglobin show (smaller) perturbations of tryp-
`tophan residues upon ligand exchange without the
`pronounced difference in response between high-spin
`and low-spin complexes. Even the bulky ligand,
`nicotinic acid, does not create a significantly larger
`perturbation to the structure than the other ligands.
`There is a high degree of structural homology be-
`tween myoglobin and leghaemoglobin revealed by
`sequence alignments [24 - 291 and the recent X-ray
`structure of lupin leghaemoglobin [30]. Pursuing this
`structural homology, neither myoglobin nor leghaemo-
`globin contain tyrosine or tryptophan residues in or
`very near to the haem pocket and this implies that
`ligand exchange and especially a high-spin to low-spin
`transition causes rather extensive structural rearrange-
`ments through the protein which involve these residues.
`These rearrangements may be more significant in
`myoglobin than in leghaemoglobin since the pertur-
`bation of the tyrosine residue is much greater than that
`of tryptophan in leghaemoglobin.
`
`139
`
`We have presented evidence elsewhere [4,8] that
`the haem group in leghaemoglobin may be in less
`intimate contact with protein side chains and more
`exposed to aqueous solvent in leghaemoglobin com-
`pared with myoglobin. The results in this paper sup-
`port this idea by showing that myoglobin structure is
`affected relatively more by spin-state changes than is
`leghaemoglobin structure. The failure of rose Bengal
`binding to increase the helicity of apomyglobin (as it
`does for leghaemoglobin) may also reflect the more
`stringent requirements for haem-protein interactions
`in myoglobin where the fit is tighter (i.e. rose Bengal
`cannot mimic the interactions provided by the haem
`in native myoglobin but can in the more flexible
`leghaemoglobin pocket).
`The concept of a more flexible, open haem pocket
`in leghaemoglobin helps to explain many of the pro-
`perties of leghaemoglobin compared with myoglobin,
`in particular its ability to bind bulky ligands and its
`high oxygen affinity. The absence of Tyr-H22 in leg-
`haemoglobin also means that a tyrosine is not trans-
`ferred from a non-polar to an aqueous environment
`(an energy-requiring process) upon oxygenation (high-
`spin to low-spin transition) further accounting for the
`higher oxygen affinity of leghaemoglobin compared
`with myoglobin.
`
`The authors are grateful to Dr C. A. Appleby (Division of
`Plant Industry, Commonwealth Scientific and Industrial Research
`Organization, Canberra) for gifts of soybean leghaemoglobin a and
`for helpful discussions. They also wish to thank Professor M. J.
`Dilworth (School of Environmental and Life Sciences, Murdoch
`University, Murdoch, W.A.) and Dr W. J. Broughton (School of
`Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia)
`for valuable gifts of serradella and lupin leghaemoglobins. This
`work was carried out with the aid of a grant from the Australian
`Research Grants Committee to S.J.L. N.A.N. was supported by a
`Commonwealth Scientific and Industrial Research Organisation
`Studentship.
`
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