Spectroscopic Studies of Cobalt and Nickel Substituted Rubredoxin and Desulforedoxin Isabel Moura, Miguel Teixeira, Jean LeGall, and Jose J. G. Mom-a IM, MT, JJGM. Centro de Tecnologia Quimica e Biol6gica and Universidade Nova de L&boa, Oeiras, Portugal.-JL. Department of Biochemistry, University of Georgia, A thens, Georgia
ABSTRACT The single iron site of rubredoxin was replaced by nickel and cobalt. The near-infrared/visible/UV spectra of these metal derivatives show ligand-field transitions and charge-transfer bands which closely resemble those of simple tetrathiolate complexes, indicating a tetrahedral arrangement of the sulfur cysteinyl ligands around the metal core. The ‘H NMR spectra of the nickel and cobalt derivatives reveal extremely low-field contact shifted resonances of one proton intensity assigned to @-CH, and wCH cysteinyl protons. Other well resolved resonances shifted out of the main protein spectral envelope are also observed and probably arise from contact plus pseudocontact shift mechanisms. Rubredoxins from different sulfate reducers were metal substituted and assignments of aliphatic protons ton>
METAL SUBSTITUTED
RUBREDOXIN
129
Cys(6)-a-b-Cys(9) and Cys(39)-c-d-Cys(42)-Gly as observed in rubredoxin is replaced by the sequences Cys(9)-x-y-Cys( 12)-Gly and Cys(28)-Cys(29)-Gly. This unusual arrangement with two adjacent cysteinyl residues may impose stereochemical constraints which are responsible for the spectroscopic differences between desulforedoxin and rubredoxin [lo]. A new non-heme iron protein isolated from D. vulgaris (Hildenborough) was designated as rubrerythrin. This protein contains two rubredoxin-like FeS, centers and a hemerythrin-like binuclear iron cluster. The physiological function of this protein with this unusual combination of prosthetic groups is unknown [21]. In this paper, we report the replacement of iron by cobalt and by nickel in rubredoxin and desulforedoxin isolated from D. gigas. Metal analysis, UV/visible near infrared, and EPR and NMR spectroscopic methods are used to characterize these metal derivatives. Replacement of iron by cobalt was previously reported for the rubredoxin of Pseudomonas oleovorans [22]. We have previously reported two studies on nickel-replaced rubredoxins [23, 241. MATERIALS
AND METHODS
The growth conditions for D. gigas and the purification of rubredoxin and desulforedoxin have been reported previously [19, 251. Rubredoxins from D. desulfuricans strains 27774 and Berre eau are additionally considered and were obtained as indicated in references 26 and 27. Unless otherwise stated, we are referring to the proteins isolated from D. gigas. Reconstitution
Procedure
of Rubredoxin
and Desulforedoxin
with Cobalt and Nickel
The protein was precipitated at 4°C with trichloracetic acid (final concentration 5%) in the presence of 0.5 M fl-mercaptoethanol. The colorless precipitate was collected by centrifugation and then dissolved in 0.5 M Tris base containing 60 mM pmercaptoethanol. The procedure was repeated and the protein was precipitated twice to avoid iron contamination. To reconstitute the protein with cobalt or nickel the apoprotein, at a concentration of about 5 mg/ml, was incubated at 25°C in argon in 0.5 M Tris base containing 60 mM P-mercaptoethanol. After 30 min incubation, cobalt(I1) or nickel(I1) nitrate was added at an equimolar concentration to the apoprotein. The mixture was allowed to react in argon for about ten min. The solutions were exposed to air and then purified on a Sephadex G-25 column (1 x 20 cm) equilibrated at pH 7.6 with 10 mM Tris HCI buffer. The rubredoxin and desulforedoxin cobalt derivatives are pale greenish and the nickel ones are yellow. The cobalt and nickel derivatives of both proteins are stable upon air exposure. Metal and Protein
Determination
Cobalt, nickel, and iron determinations were made by plasma emission spectroscopy using a Jarrell-Ash model 750 Atomcomp. Protein was determined by Lowry’s method [28] using a bovine serum albumin standard solution purchased from Sigma Chemical Co. Instrumentation The UV visible spectra were recorded on a Shimadzu Model 260 Spectrophotometer in buffered aqueous solutions (Tris-HCl). The near-infrared spectra were measured on a DS Model 14 Spectrophotometer using D,O as solvent. The contribution from the remaining water was subtracted.
130
I. Moura
et nl.
For
the NMR
studies.
(Arnicon)
apparatus
resolution
NMR
CXP
Spectrometer
300
the Ni-
and Co-derivatives
and the solvent
spectra
were
was exchanged
were
recorded
(300
MHz)
in the Fourier
quipped
with
an Aspect
was controJJetJ t(\ A o.s”C
with
a standard
temperature
control
shift
values
(ppm)
internal
and
upfisld
shifis
suppression transients a center
unit.
was WeJe
411 chemical
sodium
are
carried
as positive
iJut.
cdicctcd
OVCJ
licld at 60 kHr,
a
For
D20.
protein
2000
computer.
&-\‘I‘--
are quoted
i I)()()
in papli
band width
an
I’hc
ari:ihk
I
per million Downhrld No
rcspectlvcl!
spectrum,
High-
mode on a Hruker
l%ru~er
and ncgativc‘.
each
‘l‘hc repetition
in a Diafo
with
1 ? 3 ._3- ‘H 1 i prcyGon;ttv. (_._..
i-trimethyJsilyl
reported
times
Transform
tcmperaturc
from
concentratrd
several
;.I\ cr,ige
\o1\ent oi’ X!WO
10.38-i d;rl,t point\
of 12.5 JWY using
,md
rate used was 11.2 WC.
RESULTS Metal Content The results of the metal doxin
derivatives
six determinations. tally
electronic
the visible broad 335, for
in the ruhredoxin
and Co-Desulforedoxin
The
absorption
spectral 515,
the Co-derivative
infrared molar
region
In
transitions
Figure
compared
?A
with
from the
infrared
TABLE
elcctrctnic
2R.
In
wcrc obscrvcd
a~
(Table
I?00
and
2). Gmilar
aha,rrptivities
of
IO rhl~se observed
rubrcdoGn
1650 m
\.?3j. iirc
In the near
tdwrvcd
with
’ (d-d transitions). Xt ,150 mn a hand is their is lis5igleti tc charge of 3340 M ’ cm ’ cm
absorption
(S-qsteinyl) spectrum
for Co( JJ)-rubrcdoxi!!.
;15 in the Co(II)-rubredoxin
to rptivities 01’
bands at 448
be assigned IIS d-d tra1Gtior.s the bound
gigus
nn~ and
! CR\ ’ dYi; dcttxteci (Tahlc ligarrd cy.xtein\!
Figure 3 (left and right panels) and Figure 4 show the low-field of the 300
is shown.
at 720 nm and 671) nni. and t\vt> more in the
2). The first four bands n~ay most probably ones
Spectra
Ni-rubrcdoxin
and
the
last
resrduc>.
and high-field
region
and the temperature
18 I(). The spectra reveal eight extremely
io\v-field
shifted
METAL SUBSTITUTED
I
I
I
I
I
I
300
I
I
I 200
,
RUBREDOXIN
133
I
mm.
FIGURE 4.
300 MHz ‘H NMR spectra of D. Gigas Ni-rubredoxin
in the 380-140 ppm region. The spectra are portions of the spectra of Figure 3 and were obtained at the following temperatures: (i) 298 K; (ii) 303 K; (iii) 308 K; (iv) 313 K, and (v) 318 K.
(chemical shifts determined at 308 K), with an average line width on the order of 800 Hz. These resonances all have one-proton intensity. Other well resolved low-field (35 to 10 ppm, Fig. 3, left panel) and high-field (0 to - 28 ppm, Fig. 3, right panel) shifted resonances can be observed outside the main spectrum of the protein envelope. These peaks follow a Curie-law temperature dependence in the temperature range possible to obtain the spectra. Several resonances are identically well resolved in the very low- and very high-field spectral regions of the ‘NMR spectra of the Co-rubredoxin (Fig. 5). Seven resonances, with intensities of one proton each, are detected in the very low-field region (260.4, 257.4, 150.5, 140.3, 120.6, 110.6, and 88.9 ppm, as measured at 308 K). These resonances show a Curie law temperature dependence. A large number of resonances are also observed between 60 and 10 ppm and 0 and -50
ppm. The ‘H NMR spectra of the Ni and Co-derivatives obtained from apo-desulforedoxin also show very low-field shifted resonances. As an example (not shown), the spectra of Co-desulforedoxin has one-proton resonances at 350, 322, 276, 263, 248, 227, 112 and 100 ppm (at 298 K). EPR Spectra No EPR spectra could be observed for the nickel derivatives of both proteins. In Figure 6, the EPR spectrum of the Co-desulforedoxin is presented. The g values observed are characteristic of high-spin cobalt(I1) (S = 3/2), and similar to Co model
METAL SUBSTITUTED
RUBREDOXIN
135
distorted tetrahedral high-spin Co(I1) core. We report here for the first time the bands observed in the near infrared region. These bands between 1000 and 2000 nm correspond to the spin-allowed ligand field transition 4A2 +4T, (F). The intense band at 350 nm is very likely an S + Co(I1) charge transfer absorption. The band at 470 nm is also suggested to be a charge transfer band. This band, in the spectrum obtained for the Co-rubredoxin, has a much lower intensity than the one reported in [22], and can be bleached upon addition of small amount of dithionite. We suggest that this band does not belong to the Co-(S-cysteine), core and is probably due to unspecifically bound Co(II1) or Co(I1) (the ratio between the 350 nm and the 470 nm band varies slightly depending on the preparation). The visible spectrum of Corubredoxin is very similar to the model compound [Co(S-ET),]*and supports the indicated assignments [32]. The Co-desulforedoxin spectrum is very similar to the Co-rubredoxin one, indicating a very similar environment of the Co(I1) site. The differences observed are probably related to the sequential arrangement of the cysteinyl residues in both proteins. The native rubredoxin and desulforedoxin already presented different visible spectra reflecting a somewhat altered symmetry around the iron core [19]. The Ni-rubredoxin visible spectrum shows a high degree of similarit,y with the one of Ni(II)-substituted aspartate transcarbamoxylase [28]. This is the only other published biological example of a monomeric Ni(I1) center ligated by four cysteinyl residues. Comparison of the visible spectrum of Ni-substituted rubredoxin with the published spectra of well characterized Ni-thiolate complexes, like Ni(SPh)i[34], suggests an assignment of the bands at 448 nm and 355 nm to S + Ni(I1) charge transfer transitions and the bands at 1751, 1223, 720, and 670 nm to Ni(I1) ligand field transitions. The bands at 720 and 670 nm are assigned to transitions “T,(F) +3T, (P) and the band at 1750 and 1223 nm to transitions 3T, (F) +3A2 of Ni(I1) in an approximately tetrahedral coordination geometry [35]. Another reported four-coordinated Ni(I1) complex, containing only sulfur ligands and with a tetrahedral structure is the bis (imidotetra-methyldithiodiphosphino-S,S)nickel(II) complex [36]. The Ni-S distances in these two Ni(I1) inorganic complexes are similar but significantly longer than the ones found in square planar complexes. The angular distortion of the Ni(SPh)icomplex is more pronounced [34]. The low temperature MCD studies of D. gigas Ni substituted rubredoxin also support a tetragonally-distorted tetrahedral thiolate coordination for Ni(II), resulting in a ground state 3A2 under D *,, symmetry, that is subjected to a very large axial zero field splitting (D = 55 cm- ‘) [23]. The magnetization data obtained [33] are compatible with a spin state of S = 1 for the Ni-rubredoxin and S = 3/2 for the Co-rubredoxin. These results are in accordance with the NMR (see below) and EPR data presented here. The unusually high value determined for the axial zero field splitting precludes the observation of an EPR signal associated with the nickel S = 1 spin system. The Ni-desulforedoxin visible spectrum is quite different from the one observed for Ni-rubredoxin. The band at 375 nm and the shoulders at 405 nm and 350 nm are and the bands at 610 nm and probably charge transfer transitions from S +Ni(II) 1127 are assigned to Ni(I1) ligand field transitions. The NMR data presented for both Ni- and Co-derivatives of rubredoxin reveal a large number of resonances that are extremely shifted outside the main protein envelope spectral region (both for high and low field spectral regions).
136
1. Moura et al.
Two groups of isotropically shifted resonances in the i H NMR spectra ol Ni-rubredoxins are assigned to &CH, (eight resonances in the spectral region 370 to 150 ppmj and wCH protons (four resonances in the spectral region .iS to 14 ppml of cysteinyl ligands. The range of’ chemical shifts observed ti)r the SCH, of the Ni-rubredoxin is larger than the one observed for the ~e(Ii~.rlrbrctfox~r: 137). in this ! 50 --2h0 pD,ltl. flour resonances were assigned in the region case the &CM, resonances were observed in this spectral region corresponding to two protc~s ca$~h. The position of’ these resonances shift upfield with increasing tcmpcraturc. bhou rug a It \{a:, ,.jf’ linear dependence of the CzhcrnicaI shift on the reciprocal hv tiiflcrcnt proposed that due to the site symmetry (approxiruatcl!, I>:,, a\ ck\