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Preliminary crystallographic data for Azotobacter cytochrome c5
LETTERS ~0 THE EDITOR
Preliminary Crystallographic
Data for Azotobacter
Cytochrome c5
‘I’wo closely related crystal forms of dimeric v~toctrrome c5 from Azotobacter cvkelandii have been grown. The crystals belong to spaw groups C2 with a 15~0, b 7 38.4. c :: 41.3 ,% and j3 ~- 101 0’: atld Cl (a centered triclinic cell) wit11 a = 46.0, b =: 37.6. c = 49.4 ,k. OL-: 87” 20’. /3 98” 40’ and y = 90” 0’. In C2 the 24,000 molecular \veipht, dirner lies 011a cr~st~alloprapllic 2.fold axis: io (‘1 tllr entire dimcr occ~~pies tire as?-rnmrtrics rurit. Azotohncter vkekzndii is a nitrogen-fixing, aerobic bacterium. Respiratory rates in this organism are higher than in any known cell, reflecting the high energy requirements for nitrogen fixation and the need to detoxify 0,. Cytochrome cg is one of two major diheme cytochrome c pigments in Asotohactel (Tissi+res, 1956; Neumann & Burris. 1959). The respiratory elect)ron transport syst’em is branched and donation of electrons from cytochrome c5 to cytochromr o forms the third phosphorylation site (Yates & Jones, 1974). As a dimer purified cytochrome cg functions well in an assay with Azotobacfrr oxidase particles (Swank & Burris. 1969). However, cyeochrome (‘6 shows no cross-reaction with mammalian cytochromc oxidase, even though Azotoha&r cell-free particles arc capable of oxidizing horse heart cytochromc c (Swank & Burris, 1969). As part of a crystallographic st’udy of the electron t,ransport proteins of dzotobactr? structural analysis of cytochrome cg has been undertaken. In comparison w&h cyt’ochromes c whose structures are known (e.g. tuna cytochrome c; Swanson et al., 1977). cytochrome cg is unusual in that’ it is isolated and crystallized as a dimer. The dimer is dissociated in II M-urea or during sodium dodecyl sulfate/acrylamide gel electrophoresis. The monomer has a molecular wright of 12.000, contains one heme and its amino acid composition includes one histidine, two methionines and a large number of acidic residues (pl -~ 4.4 and 4.2 for oxidized and reduced states, respectively) (Swank & Burris, 1969). The sequence of the heme peptide. residues 10 t’o 40, contains histidine and two cysteines (Ambler. 1974). Samples for crystallization are prepared using established procedures (Swank $ Burrix. 1969 : Campbell et al.. 1973). The purit,y index, absorbance of reduced probein at 555 pm verms oxidized protein at, 270 pm, is t,vpically 1.10 to 1.20. Neumann & Burris (1959) reported small cryst,als of both oxidized and reduced cytochrome cg. Large crystals were first grown by slow evaporation of potassium phosphate/ammonium sulfate solutions (I,. C. Sicker. C. D. Stout. Y. I. Shethna and M. Sundaralingam. unpublished results). In the, present work two closely related crystal forms have been grown. a monoclinic form and a triclinic form. Both are grown using the same conditions. Droplets. 50 to t 00 ~1 in size. which contain 30 mg protein/ml, 1.2 M-(NH&SO, and 0.2 M-pot’assium phosphate at pH 6.7, are equilibrat,ed through t,he vapor phase against’ a reservoir of 2.5 “-(NH&SO, at, room temperature. The rate of crystal formation varies from one week to three mont,hs deptxnding upon t,he preparation. This may result from the multiplicit,y of amidation
in the asymmetric
density
Crystal
(A)
f l)~n~~tl
(vol. ‘y,)
from ~~recrssion films for the monorlinir
limit
Diffraction
(mm)
Sol\wlt~ content
(g/cm”)
size of crystals
unit
units in the unit ccl1
I-, (A3/daltor,)
Typical
Parameter
of monomers
uf asymmetric
Sumber
Xumhcr
of unit cell (A,])
Vollmw
1
form. and from tliffrartometer
2.0
20
1.34
setting
0.6 h 0.5 x 0.0”
1.49
1
4
71,350
angles for the triclinic
n = 45.0, b ~ 38.4, c = 41.3, fl == 101” 0’
Crystal data for cytochrom,e cj
TABLE
form.
1.7
Xl
85,050
a 7 46.02, h == 37.63, c z ‘lg.44 1 = xi0 22’, fi rz 96” 42’, y ~ go0 0”;
LETTERS
TO
THE
El)I’I’OK
107
states which can arise during isolation (Swank & Burris, 1969). In general, preparations which give two homogeneous bands (oxidized a,nd reduced) during isoelectric bands occur focusing tend to crystallize in a short t,ime, n,hereas when multiple crystIallization is very slow. These preparations are otherwise homogeneous wit’11 respect to cytochrome c5. The quickly growing cryst,al form (monoclinic form) grows as clust’ers of thin rhombic plates. These crystals apparently contain the protein in the oxidized state as there is a rapid change from red t’o a tangerine color when solutions of t,he reducing agent Na&O, are diffused in. Thin plates can also be gro\vn from (NH&SC), in acetate or Tris buffers and in the presence of a variety of other counter-ions. The slowly growing crystals (triclinic form) appear as deep red rhombic prisms up t*o 1 mm in maximum dimension, but otherwise very similar in appearance to the thinner plates. Since no precaution is taken to keel) the protein reduced, it is assumed t,hat t)hesr cryst,als also comain oxidized prot,ein. Crystallographic data are summarized in Table 1. Bot.h crystal forms diffract, \vrll and are very stable to X-ray irradiation, showing no decay after a week of exposure. To determine t(hc unit cell data for the thin rhombic plat,es. zero level and first level precession photographs as well as cone-axis photographs \\.ere t,akrn along all t,hrec principal reciprocal latt,icc dir&ions. The following exact symmetries were observed: hO1, 2: hll, 2: Olcl, mm; lkl. m: ~/CO, mm : h,kl. m. All films exhibited systematic absences when h. + k i 2~. These and the 2/m symmetry establish the space group as c’%. The Matthews coefficient I’, (Matthr\\.s. 1968) wit)11 just one 12.000 dalton mononiPr per asymmetric unit is 149 L43/tlnlt~on. Alt~hott,gh this is unusually low for a
FIG. 1. Precession phutograph of ,thc hkt) ZOIW of a t,riclinic .4. ~iueklurlii cytochrornv v5 respectively. Si-filtered C’ulir radiation, precession CryYtal. a*, b* axes vertical and horizontal, angle p = l?“, F = 60 mm,‘exposure time 16 h.
10X
c.
I). S’1’01~‘l’
protein crystal. it, is consistent with the calculated solvent cont)ent (fi :=_:0.72 ml/g) and observed density. Thus the cytochrome c5 dimer must lie on a crystallographic 2-fold axis. The packing constraints also indicate t’hat, the dimensions of the monomer are roughly 20 a :X i0 d ‘: 40 A. The tjricIinic crystals are pseudo-monoclinic. Precession photographs show the following pseudo-symmetries: hk0. mm (Fig. 1): hkl, m: W. mm: 1kl. m. A centered triclinic cell (Cl) has been chosen for convenience in comparing t,he two cryst’al forms. In the triclinic cell the dimer. rather than lying on a 2-fold axis. occupies the entire asymmetric unit. Although this doubles the size of the problem. the larger volume of the triclinic crystals makes them more suitable for dat’a collection. Using one crystal, a data set to 2.5 &&resolution (5700 independent reflections) has been collect~ed by diffractometer methods. dgreementjibetween equivalent and remeasured reflections is 50/6 and 2(:/b, respectivel,v. If monoclinic symmet’ry is imposed on t,he dat,a to 4 A. the agreement’ is 12.7~/~. reflecting t,he pseudo-svmmetry. The author thanks L. Berk for technical assistallce. Diffractomct.er programs used in data collection were written by Dr R. McCllwr. This work has been support,ed by grants from the National Institut,es of Health (GM-25672), the Health Research Services Foundation, Pittsburgh, PA (U-15), and the Dc~velopment Fluid of the Provost, University of Pittsburgh. Department University Pittsburgh, Received
of Crystallograpt of Pittsburgh Prl 15260,~U.S.A. 14 August
C. D. STOUT
11
1978
REFERENCES Ambler, R. T. (1974). Syst. Zool. 22, 554-565. Campbell, W. H., Orme-Johnson, W. H. & Burris, K. H. (1973). Rio&em. .J. 135, ti17630. Matthews, B. W. (1968). J. Nlol. Biol. 33, 491 -497. Neumann, N. P. & Burris, R. H. (1959). J. Biol. Ghem. 234, 2386-2390. Swank, R. T. & Burris, R. H. (1969). Biochim. Bi0ph.y.s. Acta, 180, 473- 489. Swanson, It., Trus, B. L., Mandel, N., Mandel, G., Kallai. 0. R. & Dickerson, K. E:. (1977). J. Biol. Chem. 252, 759.-775. Tiss&es, A. (I 956). Biochem. J. 64, 582.--589. Yates, M. G. & Jones, C. W. (1974). ArEva?~. Miwobiol. Physiol. 11, 97-135.
+ R = ZIPI
-
PII
ICY4
.
Preliminary Crystallographic
Data for Azotobacter
Cytochrome c5
‘I’wo closely related crystal forms of dimeric v~toctrrome c5 from Azotobacter cvkelandii have been grown. The crystals belong to spaw groups C2 with a 15~0, b 7 38.4. c :: 41.3 ,% and j3 ~- 101 0’: atld Cl (a centered triclinic cell) wit11 a = 46.0, b =: 37.6. c = 49.4 ,k. OL-: 87” 20’. /3 98” 40’ and y = 90” 0’. In C2 the 24,000 molecular \veipht, dirner lies 011a cr~st~alloprapllic 2.fold axis: io (‘1 tllr entire dimcr occ~~pies tire as?-rnmrtrics rurit. Azotohncter vkekzndii is a nitrogen-fixing, aerobic bacterium. Respiratory rates in this organism are higher than in any known cell, reflecting the high energy requirements for nitrogen fixation and the need to detoxify 0,. Cytochrome cg is one of two major diheme cytochrome c pigments in Asotohactel (Tissi+res, 1956; Neumann & Burris. 1959). The respiratory elect)ron transport syst’em is branched and donation of electrons from cytochrome c5 to cytochromr o forms the third phosphorylation site (Yates & Jones, 1974). As a dimer purified cytochrome cg functions well in an assay with Azotobacfrr oxidase particles (Swank & Burris. 1969). However, cyeochrome (‘6 shows no cross-reaction with mammalian cytochromc oxidase, even though Azotoha&r cell-free particles arc capable of oxidizing horse heart cytochromc c (Swank & Burris, 1969). As part of a crystallographic st’udy of the electron t,ransport proteins of dzotobactr? structural analysis of cytochrome cg has been undertaken. In comparison w&h cyt’ochromes c whose structures are known (e.g. tuna cytochrome c; Swanson et al., 1977). cytochrome cg is unusual in that’ it is isolated and crystallized as a dimer. The dimer is dissociated in II M-urea or during sodium dodecyl sulfate/acrylamide gel electrophoresis. The monomer has a molecular wright of 12.000, contains one heme and its amino acid composition includes one histidine, two methionines and a large number of acidic residues (pl -~ 4.4 and 4.2 for oxidized and reduced states, respectively) (Swank & Burris, 1969). The sequence of the heme peptide. residues 10 t’o 40, contains histidine and two cysteines (Ambler. 1974). Samples for crystallization are prepared using established procedures (Swank $ Burrix. 1969 : Campbell et al.. 1973). The purit,y index, absorbance of reduced probein at 555 pm verms oxidized protein at, 270 pm, is t,vpically 1.10 to 1.20. Neumann & Burris (1959) reported small cryst,als of both oxidized and reduced cytochrome cg. Large crystals were first grown by slow evaporation of potassium phosphate/ammonium sulfate solutions (I,. C. Sicker. C. D. Stout. Y. I. Shethna and M. Sundaralingam. unpublished results). In the, present work two closely related crystal forms have been grown. a monoclinic form and a triclinic form. Both are grown using the same conditions. Droplets. 50 to t 00 ~1 in size. which contain 30 mg protein/ml, 1.2 M-(NH&SO, and 0.2 M-pot’assium phosphate at pH 6.7, are equilibrat,ed through t,he vapor phase against’ a reservoir of 2.5 “-(NH&SO, at, room temperature. The rate of crystal formation varies from one week to three mont,hs deptxnding upon t,he preparation. This may result from the multiplicit,y of amidation
in the asymmetric
density
Crystal
(A)
f l)~n~~tl
(vol. ‘y,)
from ~~recrssion films for the monorlinir
limit
Diffraction
(mm)
Sol\wlt~ content
(g/cm”)
size of crystals
unit
units in the unit ccl1
I-, (A3/daltor,)
Typical
Parameter
of monomers
uf asymmetric
Sumber
Xumhcr
of unit cell (A,])
Vollmw
1
form. and from tliffrartometer
2.0
20
1.34
setting
0.6 h 0.5 x 0.0”
1.49
1
4
71,350
angles for the triclinic
n = 45.0, b ~ 38.4, c = 41.3, fl == 101” 0’
Crystal data for cytochrom,e cj
TABLE
form.
1.7
Xl
85,050
a 7 46.02, h == 37.63, c z ‘lg.44 1 = xi0 22’, fi rz 96” 42’, y ~ go0 0”;
LETTERS
TO
THE
El)I’I’OK
107
states which can arise during isolation (Swank & Burris, 1969). In general, preparations which give two homogeneous bands (oxidized a,nd reduced) during isoelectric bands occur focusing tend to crystallize in a short t,ime, n,hereas when multiple crystIallization is very slow. These preparations are otherwise homogeneous wit’11 respect to cytochrome c5. The quickly growing cryst,al form (monoclinic form) grows as clust’ers of thin rhombic plates. These crystals apparently contain the protein in the oxidized state as there is a rapid change from red t’o a tangerine color when solutions of t,he reducing agent Na&O, are diffused in. Thin plates can also be gro\vn from (NH&SC), in acetate or Tris buffers and in the presence of a variety of other counter-ions. The slowly growing crystals (triclinic form) appear as deep red rhombic prisms up t*o 1 mm in maximum dimension, but otherwise very similar in appearance to the thinner plates. Since no precaution is taken to keel) the protein reduced, it is assumed t,hat t)hesr cryst,als also comain oxidized prot,ein. Crystallographic data are summarized in Table 1. Bot.h crystal forms diffract, \vrll and are very stable to X-ray irradiation, showing no decay after a week of exposure. To determine t(hc unit cell data for the thin rhombic plat,es. zero level and first level precession photographs as well as cone-axis photographs \\.ere t,akrn along all t,hrec principal reciprocal latt,icc dir&ions. The following exact symmetries were observed: hO1, 2: hll, 2: Olcl, mm; lkl. m: ~/CO, mm : h,kl. m. All films exhibited systematic absences when h. + k i 2~. These and the 2/m symmetry establish the space group as c’%. The Matthews coefficient I’, (Matthr\\.s. 1968) wit)11 just one 12.000 dalton mononiPr per asymmetric unit is 149 L43/tlnlt~on. Alt~hott,gh this is unusually low for a
FIG. 1. Precession phutograph of ,thc hkt) ZOIW of a t,riclinic .4. ~iueklurlii cytochrornv v5 respectively. Si-filtered C’ulir radiation, precession CryYtal. a*, b* axes vertical and horizontal, angle p = l?“, F = 60 mm,‘exposure time 16 h.
10X
c.
I). S’1’01~‘l’
protein crystal. it, is consistent with the calculated solvent cont)ent (fi :=_:0.72 ml/g) and observed density. Thus the cytochrome c5 dimer must lie on a crystallographic 2-fold axis. The packing constraints also indicate t’hat, the dimensions of the monomer are roughly 20 a :X i0 d ‘: 40 A. The tjricIinic crystals are pseudo-monoclinic. Precession photographs show the following pseudo-symmetries: hk0. mm (Fig. 1): hkl, m: W. mm: 1kl. m. A centered triclinic cell (Cl) has been chosen for convenience in comparing t,he two cryst’al forms. In the triclinic cell the dimer. rather than lying on a 2-fold axis. occupies the entire asymmetric unit. Although this doubles the size of the problem. the larger volume of the triclinic crystals makes them more suitable for dat’a collection. Using one crystal, a data set to 2.5 &&resolution (5700 independent reflections) has been collect~ed by diffractometer methods. dgreementjibetween equivalent and remeasured reflections is 50/6 and 2(:/b, respectivel,v. If monoclinic symmet’ry is imposed on t,he dat,a to 4 A. the agreement’ is 12.7~/~. reflecting t,he pseudo-svmmetry. The author thanks L. Berk for technical assistallce. Diffractomct.er programs used in data collection were written by Dr R. McCllwr. This work has been support,ed by grants from the National Institut,es of Health (GM-25672), the Health Research Services Foundation, Pittsburgh, PA (U-15), and the Dc~velopment Fluid of the Provost, University of Pittsburgh. Department University Pittsburgh, Received
of Crystallograpt of Pittsburgh Prl 15260,~U.S.A. 14 August
C. D. STOUT
11
1978
REFERENCES Ambler, R. T. (1974). Syst. Zool. 22, 554-565. Campbell, W. H., Orme-Johnson, W. H. & Burris, K. H. (1973). Rio&em. .J. 135, ti17630. Matthews, B. W. (1968). J. Nlol. Biol. 33, 491 -497. Neumann, N. P. & Burris, R. H. (1959). J. Biol. Ghem. 234, 2386-2390. Swank, R. T. & Burris, R. H. (1969). Biochim. Bi0ph.y.s. Acta, 180, 473- 489. Swanson, It., Trus, B. L., Mandel, N., Mandel, G., Kallai. 0. R. & Dickerson, K. E:. (1977). J. Biol. Chem. 252, 759.-775. Tiss&es, A. (I 956). Biochem. J. 64, 582.--589. Yates, M. G. & Jones, C. W. (1974). ArEva?~. Miwobiol. Physiol. 11, 97-135.
+ R = ZIPI
-
PII
ICY4
.