Structural effects accompanying ligand change in crystalline lamprey hemoglobin

Biochimica et Biophysica .4cta, 31o (I973) 32-38 @3 Elsevier Scientific Put)lishing Company, A m s t e r d a m

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8BA 36411 STRUCTURAI. EFFECTS ACCOMPANYING LIGANI) ( ' t t A N G E IN C R Y S T A L L I N E LAMPREY HEMOGI.OBIN

W A Y N E A. tlI';NI)IIlCb~S()N

Laboratory/or the S'tructure of Matter, Naval Research Laboratory, Washington. D.C. 20375 ( U.N.A .) and Thomas C. ./enkins Department of t~iophysics, Johns Hopkins University, Baltimore, 31d. 21218 (17.N.."~.) (l~eceived l)ecembt'r 5th, I072)

SUMMARY

Crystals of lamprey hemoglobin in several states of ligation were prepared by intracrystalline reactions. X-ray diffraction data corresponding to a projection of the crystal structure were measured from the different ligand states: deoxy, carbonmonoxy, acid met, alkaline met, azomet and cyanomethemoglobin. These data were used to quantify the structural similarities between these ligand complexes. Structures of the low-spin complexes are all very similar, but the high-spin structures, particularly deoxyhemoglobin, differ quite considerably from the low-spin forms. The structural differences between deoxy and low-spin states are inuch greater in lamprey hemoglobin than in other monomeric globin. Bv contrast, these results are in qualitative agreement with observations on mannnalian hemoglobin. A concentration of electron density changes in the CD and FG regions of the projected difference maps between deoxy and carbonmonoxyhemoglobin lends support to the suggestion that lamprey hemoglobin dimers may be analogous to the atfl., dimers of mammalian hemoglobin.

INTRODUCTION

Hemoglobins exist in several different ligand states. Oxy and deoxyhemoglobin are those of primary physiok)gical importance ; however other states, such as methemoglobin and carbonmonoxyhemoglobin, are often more convenient to study and are also relevant to understanding the structural basis of hemoglobin action. In the case of lamprey hemoglobin, crystallization as monomers could only be achieved when using cyanomethemoglobin 1. The X-ray structure analysis of these crystals 2,3 not only showed that lamprey hemoglobin possesses the "myoglobin fold", but also provided experimental confirmation for the expectation of Hoard 4 that the iron atom should lie eoplanar with the porphyrin skeleton in a low-spin heme protein. The purpose of this report is to describe a survey of other ligand states of lamprey hemoglobin. These diffraction results help to relate the cyanomet structure to other forms of

I . I G A N D C H A N G E S IN L A M P R E Y H E M O G L O B I N

33

tile molecule and in turn to relate the structural effects of ligand change in lamprey hemoglobin to those observed in other globins. MATF.RIALS A N D M E T H O D S

Crystals of type I) 2 were grown by salting lamprey (Petromyzon marinus) cyanomethemoglobin out of solution with Drabkin's buffer (2.8 M potassium phosphates, pH 6.8) at 75% full strengtha. 3. These crystals belong to space group P212121 and have cell dimensions of a = 44-57, b = 96.62 and c -- 3z.34 3~. Native, cyanomethem(globin, crystals were converted to other ligand states by intracrystalline reactions in appropriate soaking media. The procedures fl)llowed were essentially those described previouslyk First the crystals were washed twice in cyanide-free 90°.o Drabkin's buffer. The washed crystals were reduced in zo mM dithionite, 9o°o Drabkin's buffer and then washed again to rid them of dithionite and released cyanide. Some cracking, parallel to the (ooI) planes, invariably accompanied this transition from cyanomet to deoxyhemoglobin but did not affect the quality of the diffraction pattern. Carbonmonoxyhemoglol)in crystals were prepared by replacing the N., atmosphere of the deoxy crystals with CO gas. Ferric forms were produced by reoxidizing the deoxy crystals in zo mM ferricyanide. Either IO mM NaN.~ or Io mM NaCN was included in the oxidizing medium in order to obtain azomet or cyanomet crystals. The proportions of mono- and dibasic potassium phosphates were adjusted to give a medium of pH 6. 5 for acid (aquo-) methemoglobin and one of pH 8. 9 for alkaline (hydroxy-) methemoglobin. Each of the ferric crystals was washed free of ferricyanide before use. Crystals were mounted in glass capillaries containing appropriate gaseous environments for examination by X-ray diffraction. The equivalence of diffraction patterns from these crystals with ones produced by crystals which had been identified by microspectrophotometry ~, together with visual assessments of color, gave assurance that the desired ligand states had indeed been prepared. Each ligand state retains the crystal form, D, of the native cyanomet crystals. These variously liganded crystals are isomorphous to within I °,,o in unit (:ell constantsL X-ray diffraction data were measured on a diffractometer and reduced to structure amplitudes following previously established procedures .~. The data set for each crystal comprised those 397 reflections in the hko section of the reciprocal lattice which corresponded to spacings greater than 3 -~. Data were taken from two crystals each of the deoxy, azomet, alkaline met and native cyanomet (D2) derivatives; only one crystal each was used for the carbonmonoxy, acid met and reconstituted cyanomet (Dg) data. These X-ray data have been used to assess the magnitude of structural diffi~rences between ligand states. The degree of similarity between two diffraction patterns is a measure of the similarity of the structures which produced them. A t)artic ularly good parameter for expressing the overall similarity, or rather tile dissimilarity, of two isomorphous structures is the ratio of diffraction averages, k(fI

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i(A/")21

R

ZIF?]

[P]

Here F and F ' are structure amplitudes for the two crystals and k = i F2]/[F'"! is the

34

w.A. HENI)RICKSON

scale factor relating them. The average diffraction intensity, IF21, is directly related to the scattering power of the structure since [Fel ~_ r f f , w h e r e f l is an atomic scattering factor and the sum is over all atoms in the unit cell. Likewise the mean square difference amplitude, ! ( / 1 F ? . , is, for centric reflections as are concerned here, directly dependent on the scattering power of the difference structure. Thus the ratio R is a measure of relative, or fractional, structural dissimilarity. RESULTS AND DISCUSSION

The R values for all possible pairwise comparisons of the several ligand states of lamprey hemoglobin are given in Table I. An indication of the precision of these values is afforded by the self comparisons of replicated data sets and by the natiw~ versus reconstituted cyanomet comparison. Significant differences obtain even for the most similar pair, CO v e r s u s CN. An increment of o.ooo6 in R corresponds to the value expected on adding one carbon atom to each lamprey hemoglobin monomer in these crystals. Thus, some of the R values observed here, e..g. IOO carbon-atom equivalents for the CN v e r s u s deoxy differences, must represent considerable structural rearrangement. Ligand state differences derived from the hol data of Padlan 7 on bloodworm TABLE I RELATIVE STRUCTURAl. DISSIMII.ARITY BET%VEEN LIGAND STATES

R values, [(AF)ZJ/J:"-i, are given comparing data from crystals of lamprey and (;lycera hemoglobin in several ligand states. The row and column designations of a particular element in the a r r a y of It' values identify the ligand states being compared. The spin state of each ligand form is indicated by the magnetic m o m e n t s , determined from magnetic susceptibility measurements, which arc, recorded in the last line. A magnetic m o m e n t , p in Bohr magnetons, is related to the electronic spin s, of a complex a p p r o x i m a t e l y as t* ~ 2 \/s(s + l ). The s y m b o l s CO, N v CN and CN' refer respectively to c a r b o n m o n o x y - , azomet-, native cy'anonlat- and reconstituted c y a n o m e t h e m o g l o b i n . I.amprey hemoglobin values involving deoxy, N.~, CN and alkaline met crystals arc based on averaged s t r u c t u r e amplitudes from two independent d a t a sets. The bracketed quantities are self R values for these duplicated data. Parenthetical n u m b e r s are (;h,cera hemoglobin R values given for comaprison with the l a m p r e y hemoglobin data.

R values Aeicl met

Acid met

---

Deoxv

l)eoxy

CO

Alkaline met o.o99

0.030

0.o24

0.o52

o.o3(~

(O.O21)

(O.O19)

(O,OI8)

0.0046

0.040

o.o 71

o.o()o

(o.o~4)

(O.OT~))

(O.OI4)

III

CN

CN

(O.O29)

(" O

Na

Na

(). O I' [

O " () L ( )

-

(o.ox 2)

(o.oo0)

--

0.0058

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--

(0.008)

-

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-

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o.oo23

0.o37 o.o 104

5.46""

o'"

.3.17"

2..12"

CN"

4.03"

" Measurement~s from brook lamprey (Lampetra fluviatilis) hemoglobin 5. "" Measurements from h u m a n hemoglobin 6.

0.oo3 4

LIGANI) CHANGES IN I.AMPREY HEMOGLOBIN

35

(Gl),cera dibranchiata) hemoglobin are also recorded in Table I. These Glycera and lamprey results are especially comparable since circumstances of the data are similar: both include only centric reflections out to 3 A resolution, ttle averages are over essentially the same numbers of reflections (415 v e r s u s 397) and the respective sections of data correspond to electron density projections through about tile same distances (32.8 w'rsus 31.3 A). The relative structural dissimilarities between ligand forms are correlated with the spin states of the complexes. Magnetic moments are recorded in Table I to provide a measure of the spin state of each ligand form. It is evident from the R values of Table I that the structures of the low-spin complexes of lamprey hemoglobin (azomet, cyanomet and carbonmonoxy) are all closely related. In, contrast the high-spin structures, acid met- and deoxyhemoglobin, differ quite significantly both between themselves and especially in comparison with the low-spin states. Differences between deoxyhemoglobin and the low-spin structures are particularly marked. The case of alkaline methemoglobin is somewhat special since part of the structural change which occurs on raising the pH is probably due to the titration of other groups besides the ligand. Nonetheless, it ma t' be significant that lamprey alkaline methemoglobin is closer in structure to the low-spin compounds than it is to the other high-spin forms. Some information about the location of structural changes implied by the R values in Table I can be gained, in light of the native structure, from difference electron density projections as shown in Fig. 1. The CO-CN comparison ( R - - - o . o I ) gives a difference map which shows significant features only in the vicinity of the projection of tile distal histidine. It appears that this residue has moved, perhaps to accommodate different bonding geometries for the CO and CN- ligands. Contrasting with this rather featureless map, tile difference map from the CO-deoxy comparison (R = o.o4) shows extensive change. Although these projected differences def t, specific interpretation, the concentration of prominent features at the projection of helix C and the F(; corner is suggestive. It is just this region which has been implicated, by analogy with the ligand-sensitive alfl ~ interaction in mammalian hemoglobin, as a possible contact region for the homodimer of deoxy lamprey hemoglobin2, s. Structural change would be expected at the dimer contact since ligand binding causes the deoxy dimer to dissociate. Whether movements have occurred within the heme is obscure from these maps since the heme plane is oblique to this projection. The structural effects of ligand change seen in lamprey hemoglobin differ from those observed in other globin monomers. The data on Glycera hemoglobin in Table I demonstrates this. R values for comparisons among low-spin ligand states or between deoxy and methemoglobin are of comparable magnitude for both lampre.y and Glycera hemoglobin; but in comparisons between deoxy or methemoglobin and anv of the low-spin derivatives, the lamprey R values are nmch higher than the Glycera values. As judged from difference t;ourier projections (ref. 9 and Watson, It., private comnmnication), sperm whale myoglobin behaves more like GlyceraLlo than like lamprey hemoglobin in this respect. Mean difference amplitudes reported in the three-dimensional studies of azomet ~ and deoxy myoglobin 12 bear out this conclusion. The ratios, [ [AFI]/[[F[j, for the differences of azomet versus met and deoxy versus metmyoglobin are. o.o8 and o.o 9 with an error control value of o.o5 whereas comparisons, on twodimensional data, between these derivatives of lampre.y hemoglobin give values of o.23 and o.18 with a control of similar value, o.o 5. Even considering that the expected

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Fig. I. l)itterence electron density projections. The CO-CN map at upper left is a projection of the difference in electron density between the carbonmonoxy and cyanomet ligand forms. This synthesis employed Fourier coefficients of mex(IFco! .... !FcxD exp(mex) where racy and ~lcx are the tigures-of-merit and phases from the cyanomet structure analysis a. Coefficients for the ('()l)eoxy difference synthesis at lower left differed only in replacing i./:c x[ by the appropriate structure. amplitudes [Fl)eoxy'. l'ositive contours are drawn with solid lines, negative contours are dotted and the zero contour is omitted. The contour interval is o. 3 e/.~ a. The area of projection encompasses an asymmetric unit of the structure with bounds as s h o w n on the right. The drawing at right also shows symmetry elements of the crystal, the projected molecular outline, and a schematic of the polypeptide backbone with letters designating the helical segments. The projected outline of the heine group is shown by the dashed boundaries on the maps. The iron atom position is indicated by crosses at left and by the stippled circle at right. v a l u e <>f IAt:l f r o m general reflections is less, b y a b o u t 2/~t, t h a n t h a t from reflections of a c e n t r o s y m m e t r i c zone, t h e l a m p r e y difference r a t i o s are a p p r e c i a b l y higher. T h e s t r u c t u r e of t h e m o n o m e r i c insect h e m o g l o b i n f r o m C h i r o n o m u s thu~nmi also seems less affected b y c e r t a i n l i g a n d c h a n g e s t h a n is the l a m p r e y h e m o g l o b i n s t r u c t u r e . A difference s y n t h e s i s b e t w e e n d e o x v a n d c a r b o n m o n o x y C h i r o n o m u s hemoglot)in showed s i g n i f i c a n t f e a t u r e s o n l y in t h e i m m e d i a t e v i c i n i t y of t h e h e i n e g r o u p ~a w h e r e a s the p r o j e c t e d difference m a p b e t w e e n t h e s e d e r i v a t i v e s of l a m p r e y h e m o g l o b i n (Fig. ~) s u g g e s t s n m c h m o r e p e r v a s i v e changes. W h i l e t h e results f r o m l a m p r e y h e m o g l o b i n are at v a r i a n c e w i t h d a t a from mon o m e r i c h e m o g l o b i n s , t h e y do agree q u a l i t a t i v e l y w i t h o b s e r v a t i o n s on m a m m a l i a n h e m o g l o b i n . P e r u t z has f o u n d t h a t t h e t e r t i a r y s t r u c t u r e s of d e o x v (z a n d fl s u b u n i t s differ q u i t e s u b s t a n t i a l l y f r o m t h e o x y (met) c o n f i g u r a t i o n s ~4. L a m p r e y a n d m a m m a lian h e m o g l o b i n also s h a r e the p r o p e r t y of h a v i n g n m c h lower affinity fi~r o x y g e n t h a n is possessed b y m y o g l o b i n or b y G@cera or C h i r o n o m u s h e m o g l o b i n . P e r u t z has s u g g e s t e d t h a t t h e low o x y g e n affinity of t e t r a m e r i c h e m o g l o b i n is c a u s e d p r i n c i p a l l y b y q u a t e r n a r y c o n s t r a i n t s in t h e d e o x y s t r u c t u r e w h i c h effect c o n f o r m a t i o n a l c h a n g e s w i t h i n t h e s u b u n i t s ; t h e r e b y tension is e x e r t e d at t h e hemes, i n c r e a s i n g t h e o u t - o f p l a n e d i s p l a c e m e n t s of t h e iron a t o m s a n d i m p e d i n g o x y g e n a t i o n ~5. S i m i l a r forces m a y be at p l a y in l a m p r e y h e m o g l o b i n dimers. H o w e v e r o t h e r factors m u s t also

I.IGANI) CHAN(;E.'-; IN LAMPREY HEMOGLOBIN

37

e n t e r ibr, u n l i k e h u m a n h e m o g l o b i n w h e r e i s o l a t e d s u b u n i t s h a v e h i g h a f f i n i t y , t h e o x y g e n a f f i n i t y o f l a m p r e y m o n o m e r s is still low (one t e n t h t h a t o f m y o g l o b i n ) alt h o u g h it is h i g h e r t h a n in d i m e r s ~6. W h a t e v e r is t h e c a u s e o f t h e low o x y g e n a f f i n i t y , it is c l e a r t h a t in l a m p r e y as in m a m m a l i a n h e m o g l o b i n c o n s i d e r a b l e s t r u c t u r a l change accompanies deoxygenation. ACKNOWLEDGEMENTS I t h a n k E d u a r d o P a d l a n fl)r s u p p l y i n g his d a t a o n G l y c e r a h e m o g l o b i n a n d H e r m a n W a t s o n for c o m m u n i c a t i n g his u n p u b l i s h e d m y o g l o b i n r e s u l t s . T h i s w o r k h a s also p r o f i t e d f r o m d i s c u s s i o n s w i t h E d u a r d o P a d l a n a n d W a r n e r L o v e for w h i c h I a m m o s t g r a t e f u l . S u p p o r t for t h i s w o r k h a s c o m e in p a r t f r o m a N a t i o n a l R e s e a r c h Council Postdoctoral Research Associateship and from a grant, AM2528 , from the U.S. P u b l i c H e a l t h S e r v i c e t o W a r n e r L o v e .

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