.I. Mol. Hid. (1991) 218, 583-593
Structures of Deoxy and Oxy Hemerythrin
at 2-OA Resolution
Margaret A. Holmes, Isolde Le Trong, Stewart Turley Larry C. Sieker and Ronald E. Stenkamp Department of Biological Structure University of Washington, Seattle, WA 98195, U.S.A. (Received 10 October 1990; accepted 22 Novembesr 1990) The crystallographic structure analyses of deoxy and oxy hemerythrin have been carried out at 2.0 A resolution to extend the low resolution views of the physiological forms of this oxygen-binding protein. Restrained least-squares refinement has produced molecular models giving R-values of l6+3o/o for deoxy (41,064 reflections from 10 A to 2.0 8) and 17.3 ‘$& for oxy hemerythrin (40,413 reflections from 10-OA to 2.0 A). The protein structure in each derivative
is very
similar
to that
of myohemerythrin
and
the
various
met
forms
of
hemerythrin. The binuclear complex in each derivative retains an oxygen atom bridging the two iron atoms, but the bond lengths found in deoxy hemerythrin support the idea that, in that form, the bridge is protonated, i.e. the bridging group is a hydroxyl. Dioxygen binds to the pentaco-ordinate iron atom in deoxy hemerythrin in the conversion to oxy hemerythrin. The interatomic distances are consistent with the proposed mechanism where the proton from the bridging group is transferred to the bound dioxygen, stabilizing it, in the peroxo oxidation state by forming a hydrogen bond between the peroxy group and the bridging oxygen atom.
2. Materials and Methods
1. Introduction Hemerythrin is a non-heme-iron oxygen-transport protein found in various marine worms (Sanders-Loehr, 1989). It consists of eight chemically identical subunits, each made up of 113 amino acid residues and two iron atoms (Fig. 1). The active site contains the two iron atoms, one of which is always hexaco-ordinate (Fel), the other of which or hexaco-ordinate, (Fe2) is pentaco-ordinate depending
on whether
dioxygen
two iron carboxyl and an residues
complete
The iron
of liganding
residues.
atoms are in the ferrous oxidation state in deoxy hemerythrin. Oxy hemerythrin is best described as a peroxo adduct. with the iron atoms in the ferric oxidation state. Recently, we completed the structures of met and azidomet hemerythrin refined at 1.66 a (1 A = @l nm) resolution (RI. A. Holmes & R. E. Stenkamp, unpublished results). Here we report on the structures of the physiological oxy and deoxy forms at 2.0 A resolution. These forms were studied previously at 2.2 A and 3.9 A resolution, respectively (Stenkamp et al., 1985). This study confirms and extends those earlier findings. oo‘i-2X36/91/070583-11
$03.00/0
preparation
Crystals of oxy hemerythrin were grown from a fresh 1.1 milr-protein solution in O-08 &r-Tris. HCI buffer (pH 75) at 4°C by dialysis, gradually decreasing the ionic strength to @02 rvr-Tris. HCl (pH 7.5). Crystals appeared after 3 to 4 days. To ensure the productlon of crystals of known chemical composition, if the protein solution was not freshly prepared, the protein was complekly reduced to deoxy hemerythrin, then converted to the oxg form and crystallized. The buffer solution was degassed for 30 min. argon was bubbled through the solution for a further 30 min, and diothionite was added to give a I mM solution. The protein solution was dialyzed against this oxygen-free buffer overnight. Next, the buffer solution was changed to an oxygenated solution and the ionic strength was gradually decreased t,o 001 mm-Tris. HCl (pH 7.5). Crystals of oxy hemerythrin appeared after about 10 days. Within 1 to 2 weeks of oxy crystal formation. the crystals were converted to the deoxy form. First. PO2 M-Tris . HCl (pH 7.5) oxygen-free buffer was prepared in a bubbler flask assembly by degassing for 30 min and then bubbling argon through the solution for a further 30 min. In a glovebox, dithionite was added to the buffer to a concentration of PO5 mM, and a dialysis bag or cell containing oxy crystals was placed in the oxygen-free buffer. These crystals deoxygenated within 1 day. For long-term storage the dialysis bag or cell was stored in a
or other exogenous
small molecules are bound (Fig. 2). The atoms are joined by an 0x0 bridge and the side-chains of a glutamic acid residue aspartic acid residue. Five histidine the group
(a) Crystal
583
0 1991 Academic Press Limited
M. A. Holmes et al.
584
Figure 1. Stereoscopic ribbon diagram showing the secondary and tertiary
structure
of the hemerythrin
subunit.
National Resource for Crystallography at UCSD (Xuong et al., 1985). Preparation and mounting of deoxy hemerythrin crystals for data collection was done in Seattle, WA, in an anaerobic glovebox. Care was taken to degas the solutions, mineral oil and wax used to seal the crystals in the glass capillaries, but beyond that the procedures and techniques were those used for our previous crystallographic studies. In the glovebox, metal pins attached to the capillaries containing deoxy hemerythrin crystals were inserted into rubber stoppers, which were then placed in glass vials, providing a sealed anaerobic environment for the mounted crystals. These vials were then placed in larger containers with packing material to minimize vibration of the crystals when flown to San Diego. Fresh oxy hemerythrin crystals were
greased, ground glass stoppered vial, where the crystals would remain anaerobic for months. The crystals of both deoxy and oxy hemerythrin are isomorphous with those of met and azidomet hemerythrin. The space group is P4 with 2 octamers in the unit cell. Four subunits, IA, IB, IIA and IIB (see M. A. Holmes & R. E. Stenkamp, unpublished results), make up the asymmetric unit. Cell constants for deoxy hemerythrin are a = b = 8684 A, c = 80+32 A. Those for oxy hemerythrin are a = b = 8669 A, c = 8078 A. (b) Data collection Diffraction data for 3 deoxy and 2 oxy crystals were collected using the multiwire area detector facility at the
(b)
Figure 2. The binuclear iron complexes in (a) deoxy and (b) oxy hemerythrin
in stereo.
Deoxy and Oxy Hemerythrin
585
at 2.0 A Resolution
Table 1 Rejection statistics A. For data srt I for deoxy hemerylhrin 1. Swnmary of observation inlensities by resolution shells Number of observations Shell lower limit (A)
Average obs. F*
Average red.
Average F2/o(F2)
0
with FZ/a(F2) in given range
Structures of Deoxy and Oxy Hemerythrin
at 2-OA Resolution
Margaret A. Holmes, Isolde Le Trong, Stewart Turley Larry C. Sieker and Ronald E. Stenkamp Department of Biological Structure University of Washington, Seattle, WA 98195, U.S.A. (Received 10 October 1990; accepted 22 Novembesr 1990) The crystallographic structure analyses of deoxy and oxy hemerythrin have been carried out at 2.0 A resolution to extend the low resolution views of the physiological forms of this oxygen-binding protein. Restrained least-squares refinement has produced molecular models giving R-values of l6+3o/o for deoxy (41,064 reflections from 10 A to 2.0 8) and 17.3 ‘$& for oxy hemerythrin (40,413 reflections from 10-OA to 2.0 A). The protein structure in each derivative
is very
similar
to that
of myohemerythrin
and
the
various
met
forms
of
hemerythrin. The binuclear complex in each derivative retains an oxygen atom bridging the two iron atoms, but the bond lengths found in deoxy hemerythrin support the idea that, in that form, the bridge is protonated, i.e. the bridging group is a hydroxyl. Dioxygen binds to the pentaco-ordinate iron atom in deoxy hemerythrin in the conversion to oxy hemerythrin. The interatomic distances are consistent with the proposed mechanism where the proton from the bridging group is transferred to the bound dioxygen, stabilizing it, in the peroxo oxidation state by forming a hydrogen bond between the peroxy group and the bridging oxygen atom.
2. Materials and Methods
1. Introduction Hemerythrin is a non-heme-iron oxygen-transport protein found in various marine worms (Sanders-Loehr, 1989). It consists of eight chemically identical subunits, each made up of 113 amino acid residues and two iron atoms (Fig. 1). The active site contains the two iron atoms, one of which is always hexaco-ordinate (Fel), the other of which or hexaco-ordinate, (Fe2) is pentaco-ordinate depending
on whether
dioxygen
two iron carboxyl and an residues
complete
The iron
of liganding
residues.
atoms are in the ferrous oxidation state in deoxy hemerythrin. Oxy hemerythrin is best described as a peroxo adduct. with the iron atoms in the ferric oxidation state. Recently, we completed the structures of met and azidomet hemerythrin refined at 1.66 a (1 A = @l nm) resolution (RI. A. Holmes & R. E. Stenkamp, unpublished results). Here we report on the structures of the physiological oxy and deoxy forms at 2.0 A resolution. These forms were studied previously at 2.2 A and 3.9 A resolution, respectively (Stenkamp et al., 1985). This study confirms and extends those earlier findings. oo‘i-2X36/91/070583-11
$03.00/0
preparation
Crystals of oxy hemerythrin were grown from a fresh 1.1 milr-protein solution in O-08 &r-Tris. HCI buffer (pH 75) at 4°C by dialysis, gradually decreasing the ionic strength to @02 rvr-Tris. HCl (pH 7.5). Crystals appeared after 3 to 4 days. To ensure the productlon of crystals of known chemical composition, if the protein solution was not freshly prepared, the protein was complekly reduced to deoxy hemerythrin, then converted to the oxg form and crystallized. The buffer solution was degassed for 30 min. argon was bubbled through the solution for a further 30 min, and diothionite was added to give a I mM solution. The protein solution was dialyzed against this oxygen-free buffer overnight. Next, the buffer solution was changed to an oxygenated solution and the ionic strength was gradually decreased t,o 001 mm-Tris. HCl (pH 7.5). Crystals of oxy hemerythrin appeared after about 10 days. Within 1 to 2 weeks of oxy crystal formation. the crystals were converted to the deoxy form. First. PO2 M-Tris . HCl (pH 7.5) oxygen-free buffer was prepared in a bubbler flask assembly by degassing for 30 min and then bubbling argon through the solution for a further 30 min. In a glovebox, dithionite was added to the buffer to a concentration of PO5 mM, and a dialysis bag or cell containing oxy crystals was placed in the oxygen-free buffer. These crystals deoxygenated within 1 day. For long-term storage the dialysis bag or cell was stored in a
or other exogenous
small molecules are bound (Fig. 2). The atoms are joined by an 0x0 bridge and the side-chains of a glutamic acid residue aspartic acid residue. Five histidine the group
(a) Crystal
583
0 1991 Academic Press Limited
M. A. Holmes et al.
584
Figure 1. Stereoscopic ribbon diagram showing the secondary and tertiary
structure
of the hemerythrin
subunit.
National Resource for Crystallography at UCSD (Xuong et al., 1985). Preparation and mounting of deoxy hemerythrin crystals for data collection was done in Seattle, WA, in an anaerobic glovebox. Care was taken to degas the solutions, mineral oil and wax used to seal the crystals in the glass capillaries, but beyond that the procedures and techniques were those used for our previous crystallographic studies. In the glovebox, metal pins attached to the capillaries containing deoxy hemerythrin crystals were inserted into rubber stoppers, which were then placed in glass vials, providing a sealed anaerobic environment for the mounted crystals. These vials were then placed in larger containers with packing material to minimize vibration of the crystals when flown to San Diego. Fresh oxy hemerythrin crystals were
greased, ground glass stoppered vial, where the crystals would remain anaerobic for months. The crystals of both deoxy and oxy hemerythrin are isomorphous with those of met and azidomet hemerythrin. The space group is P4 with 2 octamers in the unit cell. Four subunits, IA, IB, IIA and IIB (see M. A. Holmes & R. E. Stenkamp, unpublished results), make up the asymmetric unit. Cell constants for deoxy hemerythrin are a = b = 8684 A, c = 80+32 A. Those for oxy hemerythrin are a = b = 8669 A, c = 8078 A. (b) Data collection Diffraction data for 3 deoxy and 2 oxy crystals were collected using the multiwire area detector facility at the
(b)
Figure 2. The binuclear iron complexes in (a) deoxy and (b) oxy hemerythrin
in stereo.
Deoxy and Oxy Hemerythrin
585
at 2.0 A Resolution
Table 1 Rejection statistics A. For data srt I for deoxy hemerylhrin 1. Swnmary of observation inlensities by resolution shells Number of observations Shell lower limit (A)
Average obs. F*
Average red.
Average F2/o(F2)
0
with FZ/a(F2) in given range
