Crystal forms of lamprey hemoglobin and crystalline transitions between ligand states

J. Mol. Biol. (1968) 33, 829442

Crystal Forms of Lamprey Hemoglobin and Crystalline Transitions between Ligand States WAYNE A. HENDRIUKSON,

WARNER E. LOVE AND GEORGE C. MURRAY

Thomas C. Jenkins Department of Biophysics The Johns Hopkins University Baltimore, Maryland 21218, U.S.A. and The Marine Biological Laboratory Wooa%Hole, Massachusetts02543, U.S.A. (Received I8 October1967) Hemoglobin from the se&lamprey, Petromyzonm.crinus, has been crystallized in nine basic forms having polymeric asymmetric units containing 1, 6, 8, 10, 12 or 16 monomers. This polymorphism and further variations within some of the forms are particularly dependent on temperature, salt of crystallization and ligand state of the heme iron. Additional variations in a form result when the ligand state is changed within the crystal. All the crystal types so far acquired, whether by growth or by intracrystalline transition, have been catalogued. Crystalline transitions between ligand states tend to fall into two classes. In the first case, both the crystal lattice and the intensity distribution change only a little. The transition product is essentially isomorphous with its parent. In the second case, the lattice parameters change by as much as 10% and the diffracted intensity is distributed quite differently. Changes of the ligand state in the monomeric form of lamprey hemoglobin are always accomplished isomorphously ; only the polymeric forms can undergo non-isomorphous transitions. Changes of ligand state are thought to effect in some subtle way the mod&ation of potential polymerization sites on the monomer without appreciable alteration of the protein conformation. In crystals with monomeric asymmetric units such changes would have little effect on the diffraction pattern. However, if the affected sites were actual points of contact between the subunits of polymeric crystals then, within the constraints imposed by lattice forces, a rearrangement of the subunits with attendant changes in the diffraction pattern could ensue.

1. Introduction The cyclostomes are only remotely related to other vertebrates and this distant relationship is reflected in the unique properties of their hemoglobins. For example, hemoglobin from the sea lamprey, Petromyzon mu&us, is a mixture of six electrophoretically distinct single-heme components of molecular weight about 18,666 (Rumen t Love, 1963). In solution, oxygenated hemoglobin behaves as a monomer, but when deoxygenated, it polymerizes to form oligomers of two to four units (Rumen, 1962; Briehl, 1963). Such a labile association equilibrium appears to be peculiar to the lamprey and provides an explanation for its otherwise paradoxical possession of heme-heme interaction and a strong Bohr effect (Wald & Riggs, 1951; Antonini, 829

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Wyman, Bellelli, Rumen & Siniscalo, 1964). Lamprey hemoglobin is then of interest, both in reference to the general problem of hemoglobin evolution and in regard to its own peculiar, and perhaps prototypic, properties. The X-ray crystal structure analysis of lamprey hemoglobin undertaken in this laboratory is directed toward these problems. While attempting to prepare material suitable for the structure analysis, a variety of crystals has been produced. Most of these have polymeric asymmetric units and are thereby unattractive for X-ray analysis. Only recently has the search been rewarded with crystals of monomers. In order to gain some knowledge of the structural relations of lamprey hemoglobin in its various ligand states, ligands have been reacted with some of these crystals and the resulting crystalline transition products studied. These reactions produced additional crystal types. Each crystal type encountered, whether by direct growth or by crystalline transition, has been characterized by its method of preparation, morphology, X-ray diffraction pattern, molecular size of the asymmetric unit and ligand state of the hemoglobin.

+e(

I-e

FIQ. 1. Ligand reactions of hemoglobin

(Hb stands for hemoglobin).

The reactions of hemoglobin usedin this work are shown in Figure 1. When the heme iron is in the reduced, ferrous state without a ligand, the protein is called deoxyhemoglobin and it can bind oxygen, or more readily, carbon monoxide. When in the oxidized, ferric state, it is called met-hemoglobin and it can bind fluoride, azide and cyanide ions, with a05nity increasing in that order. Each of these ligand-protein complexes has a characteristic absorption spectrum and in these studies the ligand state was determined by microspectrophotometry of single crystals.

2. Materials (a)

Hemoglobin

preparation

and Methods and

cryatdlizution

Over the past 10 years erythrocytes have been collected, as described by Rumen & Love (1903), from lampreys which were migrating up fresh-water streams on the north shore of Cape Cod to spawn. After hemolysis by freezing and thawing, the hemoglobin was purified by salt fractionation. In some cases the individual components of the microheterogeneous hemoglobin were separated by electrophoresis. The components are called hemoglobins 1 through 6 after Rumen & Love (1963). But most crystallization experiments were performed with unseparated hemoglobin, which is upwards of 6.5% components 4 and 6. The crystals were obtained by salting-out at high ionic strength, routinely with either ammonium sulfate or potassium phosphate. The usual procedure was to add to a

CRYSTALLINE

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solution of hemoglobin (3 to 6 g/100 ml.) either a saturated solution of AS? or DB. In either case, an array of tubes was prepared in which the salt concentration was incremented in small steps through the concentration range desired. The crystals grow at 40 to 70% saturated AS and at 50 to 80% full strength DB. The percentages are calculated arbitrarily assuming the volumes of the solutions to be additive. The anaerobic crystallization of deoxy- and carboxyhemoglobin required the use of sealed, all-glass tubes and specially scrubbed gases. For handling deoxyhemoglobin, a mixture of 1% Hz. in prepurified Ne was passed over hot copper screening. The excess hydrogen combines catalytically with the contaminating oxygen to form water. The effluent gas was shown to be free of oxygen using luminescent bacteria. For handling carboxyhemoglobin, reagent grade CO was bubbled through a solution which was 1.6 YNaOH, 1.0 M-Na,S,G, and 0.3&r-sodium anthraquinone-b-sulfonate, to remove oxygen, and then through 2.3 r+r-CdCl, to remove H8S which forms as the oxygen reagent ages. (b) Ligand reactions The hemoglobin used for crystallization was either oxidized by potassium ferricyanide, K,Fe(CN),, or reduced with sodium dithionite, NazSa04, in slight molar excess. Deoxyhemoglobin was maintained in a nitrogen atmosphere or converted to carboxyhemoglobin by bubbling with CO. The cyanide complex was obtained by adding a slight molar excess of NaCN to met-hemoglobin. The crystalline transitions from deoxyhemoglobin to oxy- or carboxyhemoglobin were accomplished by flushing the capillary in which the crystal was mounted (see below) for 5 to 10 mm with either Oz or CO in which the water vapor pressure was equal to that over the mother liquor of the crystal. Carboxyhemoglobin crystals were converted to oxyhemoglobin by intermittent flushing, 6 min on and 6 min off, with 0, for 1 hr. Cyanmet-hemoglobin crystals were reduced to deoxyhemoglobin with 10 mmdithionite in 90% full strength DB. After successive washings to remove the cyanide and dithionite, they were oxidized to met-hemoglobin with 10 m-ferricyanide. The transitions of met-hemoglobin crystals to liganded complexes were accomplished by soaking them in 10 rnr+r solutions of NaF, NaNs or NaCN in DB of sufficient strength to prevent dissolution of the crystals. (c) Microspectrophotontry Optical transmission spectra of single crystals were measured. The crystals were mounted in capillaries ae for X-ray examina tion and placed in the specimen plane of the dual-beam digitally recording microspectrophotometer developed by Murray (1966). To obtain meaningful spectra over the entire visual range, each crystal was scanned several times with different neutral density filters attenuating the reference beam. Particular care was taken to eliminate scattered light by using shaped image plane stops between the specimen and the detector. Sometimes the recorded transmission spectra were converted to optical density spectra on an IBM 7094 computer. Comparison of these spectra with those given in Lemberg t Legge (1949) confirmed the ligand state. Confirming spectra were taken from all types except B1, Cat Dq, D,,, E,, G1, H, and I, (see Table 1) which gave every visual evidence of being in the chemically expected ligand states. In many cases spectra were taken on the very crystal used for X-ray diffraction photographs and often on the same crystal before and after transition from one ligand state to another. (d) X-ray and optical examination The crystals were mounted in the usual thin-walled glass capillaries. The dry capillary, sealed at one end with Cenco deKhotinsky cement, was charged with mother liquor and then the crystal. An initial seal at the other end was formed by a drop of mercury to maintain the appropriate gaseous environment during preliminary manipulations. A permanent seal of deKhotinsky cement wae applied later. Deoxy-, oxy- and carboxyhemoglobin crystals were placed in Nz, Oa and CO atmospheres, respectively, by flushing t Abbreviations used: AS, an appropriate dilution of 3.9 ~-ammonium sulfate (saturated the cold room), usually lightly buffered with Drabkin’s buffer; DB, an appropriate dilution Drabkin’s buffer, which is 2.8 n-potassium phosphate at pH 6.8 (Drabkin 1948).

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the capillary with gas delivered past the mercury by a hypodermic needle and exhausted through the meniscal gap. The water vapor pressure of the gas was set at that of the mother liquor of the crystal by bubbling it through a salt solution of the same osmolarity. X-ray diffraction patterns of crystals so mounted were photographed with Cu Ka radiation on Buerger precession cameras calibrated with PbNOs crystals. In most cases 15” precession angles were used. Resolution of the small reciprocal spacings encountered in several of the lattices was achieved by using the adjustable collimator of Love, Hendrickson, Herriott, Lattman & McCorkle (1966) and layer line screens with l-mm annular openings. The crystal faces were indexed by a comparison of the X-ray lattice dimensions with crystal angles estimated by means of a crude single-circle goniometer.

(e) Lattice determin4ztion The spacings and angles on precession photographs were measured with the Supper film measuring device. Lattice parameters and estimated errors were obtained from these data by an LGP 30 computer program based essentially on the least-squares method of Patterson & Love (1960). The computed errors gave good estimates of the precision of parameters for a given crystal, but not necessarily for all crystals of that type. To allow for this variation, presumably due to slight environmental differences of the crystals, the errors recorded in Table 1 are 3 times the estimated standard deviations due to uncertainty in the measurements. Space group assignments were made on the basis of the symmetry of the diffraction pattern, the observed systematic absences and the restriction to inversion-free space groups imposed by the handedness of protein molecules. Lattices from non-orthogonal space groups have been specified by the parameters of the Bravais reduced cells. In each case the lattice determination was based on zero-level photographs from at least two crystals. (f) Determination of Z The number of monomeric molecules per unit cell, 2, was directly determined for each crystal form by a procedure based on the X-ray molecular weight determination method of Love (1957). The volume of an individual crystal was calculated from ocular micrometer measurements of its linear dimensions. The individual crystal was then dissolved in a known volume of solvent and the optical density of the solution measured, usually at the Soret band. Using predetermined molecular extinction coefficients, the number of hemes and thereby the number of monomers in the crystal was determined. From the crystal volume, unit cell volume and number of molecules in the crystal, the number of molecules per unit cell can be easily calculated. By assuming the molecular weight of lamprey hemoglobin to be 18,200 (Lenhert, Love & Carlson, 1956) and the partial specific volume to be O-751, the value found by Svedberg & Eriksson-Quensel (1934) for another species of lamprey, the volume of a molecule of lamprey hemoglobin was calculated. Together with the 2 measurements and unit cell volumes, this permitted computation of the volume fractions of liquid of crystallization.

3. Nomenclature The crystals thus far encountered fall quite naturally into a number of categories which we call basic crystal forms. The individuals comprising a basic crystal form are called types. Each of the 40 odd lamprey hemoglobin crystal types has been assigned a capital letter subscripted by an Arabic numeral. The letter identifies the basic crystal form. Crystal types of the same basic crystal form (1) are of similar habit, (2) possess roughly the same lattice parameters and (3) have closely related or identical space groups. Crystal types, designated by the subscript, may differ in lattice constants, diffracted intensity distributions, conditions of crystallization, chemical state of constituent molecules and any other operational distinction. For example, types -4, and A, are crystals which have the same morphology, lattice constants

CRYSTALLINE

LAMPREY

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833

differing by no more than 5% and space groups which differ by only a screw axis. However, one was crystallized from a microheterogeneous mixture of carboxyhemoglobin components from AS and the other as pure deoxyhemoglobin 5 out of DB and the resulting diffraction patterns differ. Consequently, they are classed as different types of the same basic form. The names of the basic crystal forms and types are in the order of the characterization of their lattices. In some cases different crystal types produce essentially the same distributions of diffracted intensities. To simplify discussion of such relations, intensity distributions have been classified also. Each different intensity pattern has been designated by the lower case letter of its form name, subscripted by a lower case Roman numeral. 4. Basic

Crystal

Forms

To date, lamprey hemoglobin has been crystallized in nine basic crystal forms. The form which grows depends on such things as the temperature, salt of crystallization, ligand state, protein concentration and the magic of nucleation. A most unusual feature of these crystals is the variation in the multiplicity of the asymmetric units of the different forms. Asymmetric units containing 1, 6, 8, 10, 12 and 16 monomers have been found. In this section the general characteristics of the various crystal forms are discussed; particulars are recorded for each crystal type in Table 1 and typical crystal habits are illustrated in Figure 2. These results are incomplete because all the parameters influencing crystallization have not been systematically explored. For example, no serious attempts have been made to crystallize either deoxy- or cyanmet-hemoglobin

: M Y

4

x

B

A

FIG. 2. Clinographic projections of representative crystals. Cry&& of typical habit for each basic crystal form are drawn upon the Cartesian axial cross at upper left. Fackl indices should be apparent from this orientation and the descriptions of morphology in the text.

D 12

TYPe

G, 650,b G, 75% G, 75%

CN CN CN D 0 co M CN F N CN

T, T, T, T, T, T, T, G,

G, 70% DB, T, G +G G, 6OyL DB,

D co co

R+D, Ds-+D, Do+% Do-t% D,+D, D,+D,o &+DII 60:6AS,pH8,RT

AS, DB, DB,

G,GOO/,DB,

D

RT RT RT

CR

RT

RT

&-+A, &+A,, &+AII AI+.& AI+AIO

T, T, T, T, T,

co

D D

D CO D co 0 0 0 0

of or reaction

40% AS, RT 50%DB,RT,Hb5 50% DB,RT,Hb4 A4;A, 40% AS, RT AC-+& 55% DB, RT

Conditions growth transition

G, G, G, T, G, T, G,

co

Ligand state

47.13 44.51 47.07 44.66 44.48 44.69 44.66 44.60 44.61 44.52 44.43

p21212, p2,212, p21212, %%% P2,%2, p2,2121 P&2,2, %212, p21212, %2121

P2,2,2,

98.05 97.96 97.76

162.06

93.74 93.13 92.93 97.04 94.38 94.15 94.30 97.29 93.89 96.73 93.48 96.73

P2 P2 P2

B222

p212121 p2,2,2, P21%2, p212,2, %.fh% P21%% p21212, p212121 p212,21

P2,2,2 P2,2,2

p21212,

group

Space

Crystal

TABLE

1

11 14 13 19 18 20 34 27 20 42 19 35

* & * + & + f& & f !m

f f & 9 6 9 10 10 10 10 7 8 8 6

15 25 24

rt 45

f f f f f & + + f + + i

93.99 96.62 94.18 95.60 96.77 96.02 96.62 96.59 96.67 96.23 96.63

57.26 57.50 57.65

243.15

146.83 146.82 146.84 145.74 146.35 146.17 146.52 145.65 145.96 146.23 146.49 14574

types of lamprey

+ + i & f f f 5 + + &

&& &

&

+ f f f f f & & f f j, +

Unit

17 13 17 26 19 20 19 16 17 20 17

6 12 11

69

16 22 19 24 24 28 32 41 51 31 32 39

cell

30.34 31.34 30.56 31.51 31.26 31.40 31.31 31.33 31.36 31.23 31.18

92.08 93.01 92.81

87.66

84.55 80.66 80.74 74.09 83.76 83.93 83.55 74.54 83.95 74.91 83.63 74.74

c

dimensions

hemoglobin

21

12 21 18 12 16 14 24 31 26 27 19 29

& * & f f f f & f & f

4 4 4 4 4 4 4 3 4 4 6

f 13 & 23 * 18

5

f * f f f f i f f f f f

(A)

102.83 103.99 103.88

a/S/r

& 3 * 4 & 4

v

0.1344 0.1360 0.1365 0.1345 0.1332 0.1347 0.1333 0.1350 0.1335 0.1338 0.1339

0.504 0.508 0.508

3.454

1.156 I.103 I.102 1.048 1.157 1.155 1.164 1.056 1.150 1.066 1.145 1.054

>: 10-e

1 1 1 1 1 1 1 1 1 1 1

8 8 8

10

8 8 8 8 8 8 8 8 8 8 8

8

Z.

32 33 33 33 32 33 32 33 32 32 33

28 29 29

47

37 34 34 31 37 37 37 31 37 31 37 31

Vol. y0 solvent

co

CN

CN

G1

HI

11

AS, CR

AS, CR

Pl

P2,2,2 P2,2,2 P21212 P21212 P212,2 P21212 P212,2

g1

f,

81.28

96.04

96.30

76.30

9’7.98 97-66 96.68 99.28 97.60 90.06 92.41 96.93 96.94

f

f

f

20

18

16

& 12

f 20 f 26 f 67 f 30 f 32 f 26 f 27 k 36 f 27

114.66

144.43

92-18

117.29

173.07 172.07 173.69 170.62 173.60 182.02 178.11 172.69 173.69

76

f

26

& 40

& 13

f

+ 44 & 47 f 67 f 94 & 63 f 123 & 46 f 200 & 200

49.37

91.63

69.61

68.99

10 17 18 12 18 13 21 42 28

*

f

10

18

& 16

-& 13

60.66 + 6090 f 69.26 f 67.60 f 69.64 f 69.06 * 68.63 f 68.64 f 69.27 f

f

101.26

3

6 7 6

SO.08 f 11 107.74 & 7 SO.06 & 11

f f &

100.06 97.08 106.99

0.438

1.268

0.618

0.482

1.029 1.023 0.984 0.973 I*009 0.968 0.963 0.980 0.998

12

10

8

16

6 6 6 6 6 6 6 6 6

38

28

30

26

47 47 46 44 46 44 44 44 46

Abbreviations used: Z,, monomers per crystallographic asymmetric unit; D, day-; 0, oxy-; CO, carboxy-; M, met-; F, fiuoride-; N, &de-; CN, cyanide-; G, crystal type grown; T, crystal type acquired by ligand tite transition; DB, Drabkin’s buffer; AS, cold-room saturated ammonium aulfate solution: RT, room temperature; CR, cold room temperature; Hb, hemoglobin. Percentages are calculated essuming volumes to be additive. Errors on unit cell dimensions are in the last quoted digits.

G, 60% DB, 10% Hb, RT

G, 66% DB, 10% Hb, RT

G, 60%

G, 60%

M

Fl

66% DB, CR 60% AS, CR J%-+G Er+E, &a--~ J%-+Ee Es +J% 66% DB, CR 60% AS, CR

G, G, T, T, T, T, T, G, G,

M M M N F CN CN co co

El % E3 J% E6 EC3 E7 E3 EB

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in the cold room. Nevertheless, each of the four derivatives used thus far has already been found to crystallize as two or more of a total of nine distinctly different polymorphs . (a) Crystal fomz A All crystals of this basic form are well-formed orthorhombic prisms bounded by { 1011 faces and terminated by (011) domes. Contrary to the habit predicted by the Donnay-Harker principle, elongation is in the b-direction. The crystallization behavior of basic form A is the most complicated of all the forms. There are 13 types, of which six were grown and seven were obtained astransition products. The complications arise mainly through the manner in which crystal growth is influenced by the salt of crystallization. Type A,, intensity classai, grows readily from carboxyhemoglobin in AS. Nevenka M. Rumen, in this laboratory, using deoxyhemoglobin in DB, grew types A,, A, and A, from eleotrophoretically pure components5,3 and 4, respectively. Good diffraction patterns were not obtained from A, crystals, but types A, and A, produced patterns, classali, which are indistinguishable out to spacings of 3 A. Attempts to reproduce these crystals yielded instead type A,. These have a diffraction pattern, class aiV, like that of type A, crystals which grow as deoxyhemoglobin in AS. While A, and As appear to differ only in the salt of crystallization, they are further distinguished by the transition reactions they undergo as described below. Patterns a, and aiv are quite similar, but both are considerably different from intensity class a,,. Type A, best fits the description of the “reduced” lamprey hemoglobin crystals given by Greer, Perutz & Rumen (1966). (b) Crystal form B These face-centered orthorhombic crystals of deoxyhemoglobin grow in DB. Conditions for crystallization are very similar to those for form A; in fact B, hasgrown in tubes together with A, or As. In habit they are usually six-sided plates with the tabular (010) facesbounded by { lOO} and { lOI}. (c) Crystal for778C Crystals of this form have grown only in DB. Type C, is obtained by using deoxyhemoglobin at room temperature and type C, is obtained by using the carboxy complex in the cold room. They are monoclinic laths which are flattened by (100) faces and edged by (001) and { lOl}. Elongation is along the b-axis and termination of the laths is invariably irregular. The “oxy- or met-hemoglobin” crystals reported by Greer et al. (1966) are apparently of this form. (d) Crystal form D Crystals of this form are unique among all the forms of lamprey hemoglobin crystals in that they contain a single monomer in the asymmetric unit. For this reason the structure analysis of this form has been undertaken. Molecules of cyanmet-hemoglobin pack in two fundamentally different arrangements, both with P2,2,2, symmetry, and produce two types of diffraction pattern, d, and d,,. D, and D, have pattern d, and crystallize in AS and DB, respectively. D,, D,, and D, are all of intensity classd,, and grow respectively in DB, AS at pH 8, and high concentrations of potassium citrate. The prevalence of type D, over D, indicates that it is more stable, but they co-exist in the sametest tubes. These crystals are good diffractors, with the pattern extending at least as far as l-5 A spacings.

,,,

#I

,.~.,..........~................,...~”,..~...”.....~.

” /.....,..........

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.

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.

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.

CRYSTALLINE

LAMPREYHEMOGLOBIN

837

The types grown from DB are short prisms, elongated in the c-direction, having { llO} faces terminated by (021) domes. D, crystals are bounded by the same faces but tend to be more elongated and often grow as interpenetrating edifices. The prism faces of D,, are terminated by (010) pinacoids to produce a more box-like shape. (e) Crystal form E

Form E crystals of met-hemoglobin were grown in the cold room from either AS or DB. The DB type, E,, is tabular and somewhat elongated along the c-axis. The (010) faces are most extensive and terminated by (101 } faces at the ends and {I lo} faces along the edges. The AS, E,, crystals have the (010) and (101) faces extended, usually to the elimination of the others, producing a diamond-shaped plate. These morphologies are illustrated by Rumen & Love (1963). The carboxyhemoglobin types E, and E, have the samefacial development as their E, and E, counterparts. However, they do not grow as readily and tend to be very thin plates. As a result, only poor diffraction patterns were obtained. (f) Crystal form F These were the first crystals of lamprey hemoglobin to be grown in this laboratory. They were obtained by salting-out met-hemoglobin in the cold room and must be considered relatively lessstable than type E, as they have not been obtained again. These triclinic crystals are shaped like ingots. The top and base are (010) and (OiO), the principal side faces are (100) and (ilO), and slight basal bevels are formed by (110) and (iO0). Elongation is parallel to the c-axis and the ingots are usually terminated by (001) and (OOi). (g) Crystal form G Carboxyhemoglobin crystallized in this form in the cold room in AS. The crystals are broad laths elongated in the c-direction. The lath faces are {lOO}, the edges are (110) and {liO}, and the ends often irregular, but when developed are {OOI), (011) or {Oli}. There is a tendency to cleave parallel to the edges. (h) Crystal form H These crystals grow only from high concentrations of cyanmet-hemoglobin (10 to 15%) in DB. Even at that they are small and the diffraction patterns obtained fade away at 4 to 5 A spacings. They are stout prisms bounded by (110) and (101) faces and are slightly elongated in the c-direction. (i) Crystal form I The conditions for crystallization of form I are essentially the same as for form H. Although the lattice parameters could be those of a monoclinic, the diffraction pattern symmetry is clearly that of an anorthic cell. The crystal habit is tabular. Again contrary to the Donnay-Harker principle, facial development is greatest on the (001) and (OOi) faces and elongation is in the b-direction. Ends are formed by (010) and (OiO) and the edgesvary from crystal to crystal among (lOO), (TOO)and four sign permutations of (101).

5. Ligand

State Transitions

Representatives of the nine basic forms were chosenfor studies of intracrystalline hemoglobin-ligand reactions. Forms A and C were taken as examples of crystals with 64

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polymeric asymmetric units and with the hemoglobin in the ferrous state. Form E was chosen as a polymeric crystal form with hemoglobin in the ferric stat’e. Form D is the only one with a monomeric asymmetric unit and was used in ligand reactions with both ferri- and ferrohemoglobin. Ligand reactions were confirmed by microspectrophotometry in each transition. The crystalline order as revealed by the quality and extent of the diffraction pattern remained unaffected by the transitions, despite what were sometimes rather drastic changes. The “before and after” spectra shown in Figure 3 and the corresponding precession photographs in Plate I comprise the complete record of a transition experiment.

(a) Fovm 9 transitio~ls The various transition reactions carried out on form A crystals are shown schematically in Figure 4. Crystals of the different deoxyhemoglobin types, A,, A, and A,, were reacted with carbon monoxide. Although the space groups, lattice parameters, and intensity classes of A, and A, differ, the transition products, A, and A,, are essentially indistinguishable. On the other hand, A, and A, differ in the salt of crystallization but not in diffraction pattern; however, they yield strikingly different products upon reaction with carbon monoxide. The diffraction pattern of AS-grown A, crystals makes the slight change from the deoxy- pattern, a,,, t,o the carboxy- pattern, a,. A, and A, crystals grown in DB, however, change to intensity class aiii. This pattern is really quite different from either of the parent patterns, an and a,,---lattice parameters are changed by as much as 11% and t,he intensity distribution is altered markedly. (See Plate I.)

A, (CO,

AS,

Oi)

Adboxy,

DB,

aii)

A6(dCOxy,

AS,

Ci,)

Ahboxy,

DE,

ai,)

Aa(deoxy,

DE,

aiy )

co

.

co

-A+,,,)

A,(a;)

co fW,,,)

‘(“““_,,,...,

111

4. Ligand state transitions in form A crystals. The ligand state, salt of crystallization, and intensity class of each reactant and the intrnsity class of each product crystal type are given in parentheses. Intensity classes a,, a,,, a,” and a, am all quite similar while the class a,,,, appearing only in the right-hand column, is very different. FIG.

Oxygenation is difficult to study because oxyhemoglobin autoxidizes rapidly to become met-hemoglobin, and therefore less extensive results have been obtained than with carbon monoxide. Oxygenation of the AS-grown A, crystals of carboxyhemoglobin and DB-grown A, crystals of deoxyhemoglobin led t,o similar results. Either of two diffraction patterns could result under seemingly identical experimental con.ditions. In fact, the two patterns sometimes co-existed in the same photograph.

CRYSTALLINE

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HEMOGLOBIN

839

The one type of pattern, that shown by types A,, and A13, is very similar to the greatly changed aill pattern, obtained with carbon monoxide transitions. In addition, each parent crystal can produce a much less changed resultant pattern. The aV pattern of A,, is quite similar to the parent ai pattern, but there are differences. Similarly the diffraction pattern from A,, crystals is changed but slightly, if at all, from its parent pattern. In short, while the DB crystals react vigorously with carbon monoxide to produce only the altered aili pattern, they react with oxygen to yield two alternative patterns: the ali, pattern or, preferably, one showing little change. Crystals grown in AS, however, react more vigorously when exposedto oxygen rather than carbon monoxide. A comment on the color change with oxygenation of form A deoxyhemoglobin crystals is in order here. Greer et al. (1966) reported that crystals of “reduced” lamprey hemoglobin failed to change color on exposure to air, or changed only slowly. Early experiments in this laboratory gave the same impression. However, microspectrophotometry of these crystals revealed a spectral mixture of met- and oxyhemoglobin which to the eye mimics the color of deoxyhemoglobin. Since the hydroxylation of lamprey met-hemoglobin has a pK of 7.8 (Harry Rockoff, unpublished work in this laboratory) rather than 8.1 as in mammalian hemoglobin (Austin & Drabkin, 1935), at neutral pH its color is not the characteristic brown of acid met-hemoglobin; rather it approachesthe purplish-red of deoxyhemoglobin. For this and perhaps other reasons, contaminating met-hemoglobin in the parent deoxy- crystals is difficult to detect visually. When special care is taken to prevent any oxidation of deoxyhemoglobin crystals, they change color thoroughly, visibly and readily on exposure to oxygen. This experience emphasizesthe necessity for an objective determination of ligand state in hemoglobin crystals. (b) Porn C transition The only ligand reaction studied was the type C, deoxyhemoglobin transition to type C, on exposure to carbon monoxide. No noticeable change in the intensity distribution was observed, but the lattice parameters changed to those characteristic of crystals grown as the carboxy-complex, C!,. (c) B’orrn D transitions Starting with D, crystals, which grow as cyanmet-hemoglobin, transitions were made to the ligand states as displayed in Figure 5. The crystals were exposed to various reagents dissolved in 90% full strength DB. Upon reduction with dithionite the bound cyanide is releasedyielding deoxyhemoglobin. The crystals can then react with oxygen or carbon monoxide, or can be re-oxidized to met-hemoglobin with ferricyanide and subsequently bind fluoride, azide or cyanide ions.

/D6(02)

D1(CN-)----+D5(deoxy)

D9(CN-)

----L

---‘-A

D,KO)

in form

D crystals.

I

D,~(~-,~~‘“~D,,(N;) Pm.

5. Ligand

state

transitions

840

W.

A.

HENDRICKSON,

W.

E.

LOVE

AND

G.

C. MURRAY

The most notable feature in a comparison of the resulting diffraction patterns is their general over-all similarity. The greatest difference in a lattice parameter between any two types is 1*1o/o. Although the various types are virtually isomorphous, each one has a distinctive, reproducible diffraction pattern. Intensity distributions of the cyanide and deoxy- types differ more than any others, but then only moderately. Patterns from the oxy- and carboxy- derivatives show changes from deoxyhemoglobin to the extent shown in Plate II, and also differ from one another. The oxy- pattern resembles the met- pattern more than it does the deoxy- pattern. The fluoride, azide and cyanide patterns are, in that order, increasingly different from the met- pattern. Reassuringly, after the complete cycle of reduction, oxidation and recombination with cyanide, the diffraction patterns from D, crystals are indistinguishable from those of the parent D, crystals. (d) Form E transitiolas Ligand reactions were carried out on the met-hemoglobin E, crystals by transferring them to a soaking medium of full strength DB containing ligand ions. The resulting transitions are summarized in Figure 6. These crystals are 47% by volume liquid of crystallization and, not surprisingly, changes result upon soaking without ligands, whereas the harder D, crystals do not show such changes. The soaked crystals, E,, have lattice parameters which differ by as much as2% from thenative crystals, but the intensity distribution, despite a number of changes,remains quite similar to that of E,.

E, (met)

FIG.

loo’/.

6. Ligand

strtte

transitions

in form

E crystals.

Addition of the fluoride ion to the soaking medium leaves the crystals with essentially the samelattice parameters as E, and relatively small changesin intensities. However, binding the azide ion leads to an extensive, though orderly, re-arrangement as is testified by the comparison in Plate III. Finally, binding cyanide results in two alternative lattices, E, and E,. Both are quite different from E,--lattice parameters change by as much as 6 and 3%, respectively-but the E, pattern showsmuch less correlation to the E, pattern than does that of E,.

6. Discussion The most enigmatic feature of the array of lamprey hemoglobin crystal forms is the prevalence of polymeric asymmetric units. These lack uniformity in degree of association and bear little relation to the polymerization behavior of the hemoglobin in solution. In solution, at low salt concentrations, met-, carboxy- and oxyhemoglobin all behave as monomers; polymerization has been observed only with deoxyhemoglobin and then, at most, to form tetramers. However, crystal forms occur with 6, 8, 10, 12 or 16 monomersin the crystallographic asymmetric unit, and are distributed without apparent regard to ligand state. (It should be remembered that the crystallographic asymmetric unit may, in fact, be symmetric.) Only the cyanide complex

II. Superposed precession to the right of the parent

photographs D, (deoxy,

II

dlii)

of the deoxyand oxypattern. The precession

E, (met,

III

er) pattern

above

pattern is shown

diffraction

PLATE transition products of form D. The angle is 15’ and the net is 0122.

PLATE III. Precession photographs of the form E met to azide transition reaction. The parent E, (azide, e,rl) pattern is shown below. The precession angle is 15’ and the net is Okl.

PLATE displaced

PLATE

(oxy,

d,,) and the product

of D,

is

CRYSTALLINE

LAMPREY

HEMOGLOBIN

841

has been crystallized as a monomer, and at high protein concentrations even it grows in associated forms. These results attest to the labile association behavior of lamprey hemoglobin and suggest that it is dependent on hemoglobin and salt concentrations. The crystallization behavior of lamprey hemoglobin is unique among the hemoglobins and myoglobins studied, as might be expected from the apparent lability of its quaternary structure. In the cases of sperm whale myoglobin and hemoglobin from the marine annelid, Glycera dibranchiata, both of which are monomeric, all ligand states of the hemoprotein crystallize isomorphously (Nobbs, Watson & Kendrew, 1966; Padlan & Love, 1967). Alternatively, the met-, oxy- and carboxyhemoglobin of mammals thus far crystallize isomorphously, but deoxyhemoglobin crystallizes quite differently (Bragg & Perutz, 1952). Lamprey hemoglobin behaves in neither of these manners. In some cases lamprey hemoglobin in different ligand states crystallizes in the same basic form, namely deoxy- and carboxy- in forms A and C and met- and carboxy- in form E. But it appears that other hemoglobin complexes are unable to crystallize in these lattices. Other crystal forms have been produced from hemoglobinsof only one complex despite attempts with other derivatives under similar conditions. For example, attempts to grow crystals of the azide and fluoride complexes under a wide range of conditions thus far have produced nothing suitable for X-ray examination. Further unlike the other hemoproteins, lamprey hemoglobin sometimes crystallizes in quite different types of the same basic form. This occurs not only in the polymeric forms as with the deoxyhemoglobin types A, and As, but also with the monomeric cyanmet-hemoglobin types D, and D,. Although the study of crystalline transitions of ligand state appears to complicat,e matters further, in fact it leadsto someinsight. When crystals of the polymeric forms are reacted with ligands, sometimesthe changesare slight; at other times the diffraction patterns are changed to an extent indicating rather radical rearrangement of the contents of the unit cell. Interestingly, these latter states seemto be inaccessible by crystal growth and attainable only by crystalline transition. The magnitude of these changesis not unlike that following oxygenation of reduced horse hemoglobin, where a 7% change occurs in the lattice dimension parallel to the direction of p-chain movement (Perutz, Bolton, Diamond, Muirhead t Watson, 1964). On the other hand, monomeric form D crystals change only a little after various ligand reactions. The reacted crystals are essentially isomorphous to their unreacted predecessorsand have diffraction patterns which are altered very little. These changesare like those observed in myoglobin where, as with lamprey hemoglobin, the most eccentric derivative is the cyanide complex (Watson & Chance, 1966). But even it is not structurslly far different from the others. Thus, transition studies on monomeric form D crystals demonstrate that the tertiary structure of lamprey hemoglobin is very nearly the same in all its ligand states; yet when the same ligand reactions are performed on crystals with polymeric asymmetric units, very dramatic structural changesoften occur. The most attractive explanation for this behavior is to supposethat when a ligand binds to the hemewhich for chemical reasons,in lamprey hemoglobin as well as myoglobin, is probably pocketed among the non-polar residuesin the molecular interior-structural changes are somehow communicated to polymerization sites on the surface of the monomers, but without serious distortion of the monomeric conformation, Subtle changes so induced on the surface could then affect the subunit interactions in the polymeric forms to the extent that a re-arrangement of the monomers would ensue. The con-

842

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HENDRICKSON,

W.

E.

LOVE

AND

G.

C. MUIZRAAY

siderable change in quaternary structure without appreciable tertiary alteration which accompanies oxygenation of mammalian deoxyhemoglobin (Perutz et al., 1964) exemplifies this situation. One mode of communication from the heme to the surface has been observed in myoglobin, where the reduction of met-myoglobin or binding of ions such as azide results in the expulsion, mediated by proton shifts on the distal histidine, of a sulfate ion 9 A away (Stryer, Kendrew & Watson, 1964). Whatever the particular mechanism, this sort of scheme seems a likely rationale for the behavior of lamprey hemoglobin crystals. It may also be important in the depolymerization in solution of lamprey deoxyhemoglobin upon oxygenation and perhaps even more widely in allosteric transitions in general (Monod, Wyman & Changeux, 1965). We thank the following for their helpful participation in this work : Nevenka M. Rumen, Margaret A. James, John Waterman, Frederick C. Haynes, Jon R. Herriott, Peter A. Klock, Eaton E. Lattman, Eduardo A. Padlan, Harry Rockoff, Carl Schneider and Steven Weist, who have aided our study of lamprey hemoglobin at various times over the past. ten years. This work has been supported by a grant, AM02528, from the National Institute of Arthritis and Metabolic Diseases, and by a predoctoral fellowship from the U.S. Public> Health Service to one of us (W. A. H.). The spectrophotometry was supported by a grant. GB2705, from the National Science Foundation and a grant, NB03582, from the Nat,ional Institute of Neurological Diseases and Blindness. One of us (W.A.H.) has submitted this work to t,ho Johns Hopkins University in partial fulfilment of the requirements for the Ph.D. degree.

REFERENCES Antonini, E., Wyman, J., Bellelli, L., Rumen, N. & Siniscalo, M. (1964). drclr. Bioc7,clt,. Biophys. 105, 404. Austin, J. H. 8~ Drabkin, D. L. (1935). J. Biol. Cltem. 112, 67. Bragg, W. L. & Perutz, M. F. (1952). Acta Cry& 5, 323. Briehl, R. W. (1963). J. Biol. Chem. 238, 2361. Drabkin, D. L. (1946). J. Biol. Chem. 164, 703. Greer, J., Perutz, M. F. & Rumen, N. (1966). J. Mol. BioZ. 18, 547. Lemberg, R. & Legge, J. W. (1949). Hematin Compounds and Bile Pigments. Sew York: Interscience. Lenhert, P. G., Love, W. E. & Carlson, F. D. (1956). BioZ. Bull., Woods Hole, 111, 293. Leve, W. E. (1957). Biochim. biophys. Acta, 23, 465. Love, W. E., Hendrickson, W. A., Herriott’, J. R., Lattman, E. E. 8.~ McCorkle, G. L. (1965). Rev. Sci. In&r. 36, 1655. Monod, J., Wyman, J. & Changeux, J. P. (1965). J. Mol. BioZ. 12, 88. Murray, G. C. (1966). Science, 154, 1182. Nobbs, C. L., Watson, H. C. bi Kendrew, J. C. (1966). Nature, 209, 339. Padlan, E. A. & Love, W. E. (1967). Biophys. J. 7, Abstracts 25. Patterson, A. L. &Z Love, W. E. (1960). Amer. Mineral. 45, 325. Pertuz, M. F., Bolton, W., Diamond, R., Muirhead, H. & Watson, H. C. (1964). Nature, 203, 687. Rumen, N. (1962). In Conference on Hemoglobin, spons. by Dept. of Med., Columbia Univ., p. 54. Harriman, N.Y. : Arden House. Rumen, N. M. & Love, W. E. (1963). Arch. Biochem. Biophy/s. 103, 24. Stryer, L., Kendrew, J. C. & Watson, H. C. (1964). J. Mol. BioZ. 8, 96. Svedberg, T. & Eriksson-Quensel, I-B. (1934). J. Amer. Chem. Sot. 56, 1700. Wald, G. & Riggs, A. (1951). J. Qen. PhysioZ. 35, 45. Watson, H. C. & Chance, B. (1966). In Hemes and Hemoproteirw, ed. by B. Chance, R. W. Estabrook & T. Yonetani, p. 149. New York: Academic Press.