Biochimica et Bioptwsica Acta, 1079 ( 199 ! I 161 - 168 © 1991 Elsevier Szience Publishers B.V. All rights reserved 0167-4838/91/$03.50 A D O N I S 0167483~,9100267X

161

B B A P R O 33975

Magnetization of l ,anganese superoxide dismutase from Thermus thermophilus Jim Peterson

t J a m e s A . F e e -' a n d E d m u n d

P. D a y ~

t Department of Chemtstry, Umcersitv o f Alabama. Tuscah)osa, A L (U.S.A.). " Los A/amos National Laboratory. Los Alamos. NAI (U.S.A.) and '~ Department o f Physics, Erupt)" Unicersio', Atlanta. GA (U.S.A.) (Received 20 February 1991)

Key words: Manganese superoxide dismutase: EPR; SQUID: (T. thermophilug)

The ground state magnetic properties of manganese superoxide dismutase from Thermus t h e r m o p h i l u s in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly cneeuntered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(lll) (S = 2) with D = +2.44(5) cm -n and E / D = 0. The reduced enzyme contains high spin Mn(ll) (S = 5 / 2 ) with D = +0.50(5) c m - I and E / D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(lll) present. The result, ~ 0 = 950(80) M -~ cm -~ I/t~4s o = 740(60) M - 1 c m - i ) agrees with the previously reported value of ~4so = 910 M -n c m - t found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, ! |65-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed.

Introduction

The manganese superoxide dismutase (MnSOD) from Thermus thermophilus is tetrameric ( M r = 84 000) and has on average 2.2 manganese ions bound per tetramer as isolated [1]. X-ray crystallographic data at 0.24 nm resolution indicates equivalent, mononuclear, metal ion coordination sites, with four protein ligands and possibly a water molecule, in a roughly trigonal bipyrimidal arrangement [2]. In keeping with native M n S O D ' s from a variety of sources, the isolated metalIoprotein is characterized by a distinct pink color. It was originally proposed by Keele et al. [3] that the

Abbreviations: CD, circular dichroi~m; EDTA, ethylenediaminetetraacetie acid; EPR, electron paramagnetic resonance; FeSOD, iron superoxide dismutase: Mes, 2(N-morpholino)ethanesulfonic acid; MnSOD, manganese superoxide dismutase: NMR, nuclear magnetic resonance; SDS, sodium dodecyl sulfate; SQUID, superconducting q u a n t u m inlerference device. Correspondence: J. Peterson, Department of Chemistry, University of Alabama, Tuscaloosa, AL 35487-1)336, U.S.A.

pink chromophore was Mn(ill) due to the apparent similarity of the absorption spectrum of the Escherichia coil enzyme to those of known Mn(llI) complexes [4,5]. Additional support (or this suggestion was found in the observed electrochemistry of this MnSOD [6] and the appearance of a broad Mn(ll) E P R signal, not attributable to contaminants, upon the addition of reductant [7]. Furthcrmore, the relaxation rates of manganese-bound water protons determined by N M R spectroscopy [8], the observed shift in the X-ray absorption edge upon reduction [9} and the results of two previous magnetic susceptibility studies [7 and 10] appear to be consistent with the Mn(lll) assignment in the native enzyme. Unfortunately, there remain a number of difficulties in the interpretation of this body of evidence. The near-infrared absorption bands of known Mn(lll) complexes [4,5] have not been detected in protein samples. The electrochemically determined metal-to-protein ratio [6] is in disagreement with that found by metal analysis [3] for the Escherichia coil enzyme. Mn(ll) EPR signals appear simply upon allowing MnSOD to stand at room temperature a n d / o r denaturing the

162 prote;n, seemingly in the abscncc of reducing agents [7,11]. Analysis of the N M R data concerning relaxation rates of metal-bound water protons was based on the assumption that Mn(lll} was present [8]. Finally. neither of the previous susceptibility studies were performed with entirely satisfactory samples. Morgenstern-Badarau et al. [t0j used a lyophilizcd powdcr and assumed lin common with the majority of other authors) that the manganese prescnt was I(10c¢~-, high-spin Mn(lil) in the fitting of their data. The earlier work of Fee et al. [7] shows this to have been a most unreasonable assumption. These authors had previously reported substantial amounts U Mn(ll) in their samples, which precluded quantitative fitting of magnetic susceptibility data. The question of the ground state electronic and magnetic properties of the manganese in native MnSOD and its derivatives is an important onc, since this puts constraints on possible catalytic mechanisms of the cnzyme. Given the number of independent lines of evidence in the available literature suggesting M n S O D ' s to contain high spin Mn(IIl) as isolated, we did not expect to find otherwise and indeed, now present entirely unambiguous evidence confirming this for the enzyme from Thermus thermophilus. Moreover, using combined E P R and saturation magnetization measurements, we have reliably determined the zero field splitting of the manganese in the native and reduced forms of this particular metalloprotein. When compared with the available data for model compounds, these present results show that the magnetic properties of the manganese in M n S O D are not encompassed by the properties of currently known synthetic complexes. It became apparent during the course of this study that there was considerable uncertainty in the accuracy of the reported visible region extinction coefficients for these enzymes. The extent to which the presence of unsuspected Mn(ll) in some preparations might lead to inaccurately low estimates was of prime concern. Therefore, using saturation magnetization curves to determine Mn(lll) content (rather than metal analysis to determine total manganese) we have redetermined the extinction coefficient at 480 nm for the M n S O D from Thermus thermophilus. The possible reasons for the significant variation in the visible region extinction coefficients found for M n S O D ' s from different sources are considered.

Experimental procedures Sample preparation. M n S O D was prepared from Thermus thermophilus following the previously published procedures of Sato and Nakazawa [l]. Deuteration of purified M n S O D was accomplished with the aid of an Amicon ultrafiltration apparatus and four volume

cxchanges of buffer to prepare concentrated enzyme solutions in 20 mM Mes p D 6,4 (where p D = meter reading ± 0.4 units) 2 mM in sodium E D T A . Solutions which had been subjected to this minimum buffer exchange were used for determination of ,~×tinction coefficients. In order to ensure low levels of adventitious Mn(ll) and Fe(III), samples intended primarily for use in magnetization studies were subjected to additional exchanges of deuterated buffer until they became approx. 15% depleted in m,~nganese as judged by the increase in A2sII/A480. Magnetization controls and absorption spectroscopy blanks were drawn from the filtrate of th~ final concentration step following buffer exchange. Either d6-ethanediol or ds-glycerol (ICN Biomedicals) was added to samples (10% v / v ) as a cryoprotectant. Parallel EPR, magnetization and absorption spectroscopy samples were taken from the same stock enzyme solution. Magnetizatior, samples and controls were degassed by at least three cycles of freezing, evacuation (rotary pump) and thawing, then transferred anaerobically to suprasil quartz sample holders (prepered as previously described [12])before being frozen for storage or use. Anaerobic manipulations were performed under argon using syringes and vessels closed with rubber septums. Syringe needles (Hamilton RN type - disposable) were stainless steel, previously unused, washed ( M / 1 0 E D T A solution, deionized Water, methanol) and dried in vacuo immediately prior to use. Samples were reduced by adding small quantities of reducing agent solutions to samples already in h o l d e r s / t u b e s / cuvettes. At no time were protein solutions containing reducing agents drawn through syringe needles, since this always resulted in the appearance of increased amounts of paramagnetie contaminant(s) in samples (but ncver in protein-free controls). Instrumentation. Electronic absorption spectra were measured with a Beckman DU-70 spectrophotometer. E P R spectra were obtained using a Varian E-line Century spectrometer and an Oxford Instruments E P R 910 cryostat. Magnetization data were collected using a Q u a n t u m Design S Q U I D m a g n e t o m e t e r modified as previously described [13]. At all t e m p e r a t u r e s above 8 K the sample space was backfilled four times with helium gas followed by reevacuation before allowing the samples to equilibrate prior to taking the data point. Half the data points were collected consecutively upon cooling followed by alternate points collected upon warming. This procedure exaggerates the appearance of hysteresis (due to protons for example [12]). The fact that the data shown in Figs. 3 and 4 lie on smooth curves rather than having alternate points offset on two different 'tracks' indicates the absence of hysteresis. Fittbtg magnetization data. Theoretical magnetization curves were calculated from the appropriate spin

163 A

1.0 z~ "7 E

t.J

~

~E E

0.5

B

1.0 v e"

IE

0.5 E

l

I

I

4O0

500

600

7OO

Wavetengt h (nm) Fig. 1. Electronic spectra of" Thermus thermophilus MnSOD at pD 6.4, 10 mm pathlengths, 20 ° C. (A) Native enzyme absorption spectrum (reference: buffer), 0.33 mM in enzymatic Mn(lll). {B) Difference spectrum (native minus reduced}. 11.22 mM in enzymatic Mn(lll, !1), 40-fold excess of hydrogen peroxide in the reference. H a m i l t o n i a n as d e s c r i b e d p r e v i o u s l y [12]. T h e m a g n e t i z a t i o n d a t a o f Figs. 3 a n d 4 w e r e fit by a corttputer p r o g r a m using t h e N e l d e r - M e a d downhill simplex m e t h o d [14]. T h e e r r o r function b e i n g m i n i m i z e d was s c a l e d by dividing by t h e field in o r d e r to e q u a l i z e t h e full scale c o n t r i b u t i o n at e a c h field to the e r r o r (susceptibility scaling). F o r s p e c i f i e d spin, the p r o g r a m v a r i e d ,ne spin c o n c e n t r a t i o n , z e r o field splitting p a r a m e t e r ( D ) a n d t h e i n t e r c e p t s o f t h e d a t a at each o f t h e f o u r fields. T h e g t e n s o r was set to 2. F o r native M n S O D (Fig. 3) t h e spin was set to S = 2 a n d E / D set to e i t h e r 0 o r 1 / 3 . F o r r e d u c e d M n S O D (Fig. 4) t h e spin was set to S = 5 / 2 a n d E / D set to 0.027 (from t h e E P R signal [9]). T h i s r e s u l t e d in a six p a r a m e t e r fit in e a c h case. Results

T h e visible r e g i o n e l e c t r o n i c a b s o r p t i o n s p e c t r u m o f native M n S O D shown in Fig. I A is identical to the p u b l i s h e d s p e c t r u m for this m e t a l l o p r o t e i n [1] a n d very

similar to t h o s e o f a n u m b e r of o t h e r microbial MnS O D ' s [3,11,15-18] a n d o t h e r ( m i t o c h o n d r i a l ) MnS O D ' s , including h u m a n liver [19-21]. i T h e r e were some variations b e t w e e n the s p e c t r a of p r e s e n t sampies, most n o t a b l y in t h e d c p t h of the m i n i m u m at 3q0 nm a n d in thc ! ; r o m i n e n c e of the s h o u l d e r at 410 nm. Since all s a m p l e s were p r e p a r e d in the s a m e m e d i a and c e n t r i f u g e d to remove p a r t i c u l a t e m a t t e r p r i o r to r e c o r d i n g d a t a , t h e s e s p e c t r a l d i f f e r e n c e s a r e likely due to c h a n g e s in the ratio of h a l o p r o t e i n to a p o p r o t e i n b e t w e e n t h e c o n c e n t r a t e d p r o t e i n s o l u t i o n s u s e d in these e x p e r i m e n t s . As r e d u c e d M n S O D is colorless, such d i s t o r t i o n o f the o b s e r v e d spectral c h a r a c t e r i s t i c s can b e o v e r c o m e by r e c o r d i n g the a b s o r p t i o n difference d a t a (native minus h y d r o g e n p e r o x i d e r e d u c e d ) (Fig. IB). T h e 410 nm s h o u l d is not a p p a r e n t a n d the intensity o f the b r o a d b a n d falls to z e r o at 740 nm in the d i f f e r e n c e s p e c t r u m . This is similar to the published d i f f e r e n c e s p e c t r u m for M n S O D from Escherichia coil [7]. T h e E P R s p e c t r u m of the native Thermus thermophih~s M n S O D lacks signals assignable to the active site m a n g a n e s e ( d a t a not shown). T h i s s p e c t r u m did c o n t a i n w e a k signals c o r r e s p o n d i n g to a d v e n t i t i o u s F e ( l l l ) ( g = 4 . 3 ) and M n ( l l ) ( g = 2) species. U s i n g F e ( l I I ) - E D T A a n d h e x a q u o Mn(11) as i n t e g r a t i o n stand a r d s , the a m o u n t s o f these c o n t a m i n a n t s were estim a t e d to b e 2 % ( t o t a l ) relative to t h e e n z y m a t i c m a n g a n e s e . T h e E P R s p e c t r u m o f the h y d r o g e n p e r o x i d e r e d u c e d e n z y m e is shown in Fig. 2. T h e use o f this r e d u c t a n t results in a s p e c t r u m exhibiting M n ( l l ) feat u r e s qualitatively a n d quantitatively similar to that o b t a i n e d if s o d i u m d i t h i o n i t e is used. in a d d i t i o n however, a small signal at g = 2 a n d the g = 4.3 signal o f i m p u r i t y ~ e ( l l l ) persists. T h e b r o k e n line sections o f the s p e c t r u m in Fig. 2 i n d i c a t e w h e r e the g = 4.3 and g = 2 c o n t a m i n a n t signals have b e e n s u b t r a c t e d from the data. This s p e c t r u m is similar to that previously r e p o r t e d for the r e d u c e d M n S O D from Escherichia coil [7]. T h e r e a r e two key p o i n t s to note r e g a r d i n g the E P R data. Firstly, at n o t i m e did we o b s e r v e t h e p r e s e n c e o f any r e d u c e d e n z y m a t i c m a n g a n e s e in native samples. T h i s is i m p o r t a n t b e c a u s e o f the known t e n d e n c y o f s o m e M n S O D ' s to b e c o m e a u t o r e d u c e d d u r i n g m a n i p ulation. H o w e v e r , in o u r hands, Thermus thermophilus M n S O D always a p p e a r e d to be in its fully o x i d i z e d (native) f o r m in the a b s e n c e o f a d d e d r e d u c i n g agents. T h e E P R m e a s u r e m e n t s c e r t a i n l y c o n f i r m t h e native

MnSOD's have been reported from a number of other sources: bacteria, mitochondria, algae, fungi and chloroplasts. However, we consider here only well documented examples of MnSOD's for which the extinction coefficient in the visible region, metal content and specific activity have all been published.

164 g-value

cations, it could not be used in the p r e p a r a t i o n of magnetization samples as one of the products of the reaction is molecular oxygen, which would interfere with the m e a s u r e m e n t if incompletely removed. Consequently, sodium dithionite was used to p r e p a r e reduced M n S O D for magnetization purposes. The magnetization difference data (sample minus control) of native M n S O D taken at t e m p e r a t u r e s between 1.9 and 20(1 K at magnetic fields of 0.5, 1.0, 2.0 and 4.0 tesla are p r e s e n t e d in Fig. 3A. The forth of this nested family of curves can be fit using a spin S = 2 spin Hamiltonian and cannot be fit using any o t h e r spin state "~. Therefore, the data pres,mted in Fig. 3A confirm M n ( l l l ) as the oxidation state of the metal center in the native M n S O D from Thermus ther-

)

Magnetic Field (rnTl

mophihts.

Fig. 2. E P R s p e c t r u m of r e d u c e d Tt,ermu~" thcrmophih~x M n S O D at p D 6.4. 1 m M in c n ~ m a t i c M n ( | l ) . 10 m M in h y d r o g e n peroxide. R e c o r d i n g conditions: 10 G m o d u l a t i o n a m p l i t u d e , 8. I~) ~ receiver gain. (1.2 m W microwave power, 2.5 K. 3 m m I D s a m p l e tube at X-band.

samples to contain at least 95% of the total enzymatic manganese in an E P R silent form. Secondly, the E P R spectra quite conclusively d e m o n s t r a t e that the native form is quantitatit'ely converted to the r e d u c e d form by the add;~tion of either sodium dithionite or hydrogen peroxide. Unfortunately, while hydrogen peroxide may be the reductant of choice for most experimental appli-

0.8

O

~.

Theoretical fits to the d a t a of Fig. 3A were calculated as previously described [12] using the concentration of the spin S = 2 ground state, the zero field splitting p a r a m e t e r D, and the intercepts at each field as free parameters. The data sets at the three highest fields are not very sensitive to E/D. R e a s o n a b l e fits were found at either extreme in E/D: with E / D set to zero, D was found to be +2.44(5) c m - n ; with E / D set to 1/3, D wa'.~ found to be 1.75 c m - J . The axial case ( D = +2.44 cm-~ and E / D = 0) is clearly a superior fit when these two alternatives are c o m p a r e d with d a t a at the loft field of 0.3125 tesla (Fig. 3B). F r o m this

-

0,6

/

f

fJ

#/

T IK)

,00~0

4

~ 2 o ,~ :2s

-

-

to-

_-;

,

" /

!

0.2

20

40 60 80 103/"/" (103 K ' I }

I

I

I

I

I

I

0.2

0.4

0.6

0.8

1.0

1.2

(3 ~./(kT)

"

20

4O

100

60

80

100

13, H/(RT)

Fig. 3. (A) M a g n e t i z a t i o n d a t a for native M n S O D from Thermus thermophih~s, pD 6.4: 1.9-2(IO K. A c o n t r i b u t i o n o f 2f~" (relative to m a n g a n e s e ) of an S = 5 / 2 ( B r i l i o u i n function) impurity has b e e n s u b t r a c t e d from the data. D a t a are at a p p l i e d fields of 4.0 (o), 2.0 (©), 1.0 ( A ) a n d 0.5 ( ,', ) lesla. For clarity, more t h a n h a l f of the d a t a p o i n t s have b e e n r e m o v e d from the high t e m p e r a t u r e region. T h i s e d i t i n g has b e e n d o n e w i t h o u t c h a n g i n g the a p p a r e n t s c a t t e r in the d a t a . T h e solid lines w e r e c a l c u l a t e d from a spin S = 2 spin H a m i h o n i a n with D = 2.44 c m n, E / D = 0, g = 2 for 47.1 nmol (0.150 ml). I n s e t ( A ) All of the high t e m p e r a t u r e d a t a have b e e n p l o t t e d as s u s c e p t i b i l i t y against inverse t e m p e r a t u r e . F o r clarity, the d a t a have b e e n offset by an a d d i t i o n a l 20 n J / ( T e s a m p l e ) in going to e a c h lower field. T h e lines indicate the fit at e a c h field u s e d to d e t e r m i n e the i n t e r c e p t which was set to z e r o in the main figure. (B) 41.5 nmol (0.15 m[. 0.28 m M ) of e n z y m a t i c M n ( l l l ) at 0.3125 tesla ( • ). T h e .~lid line was c a l c u l a t e d from a spin S = 2 spin H a m i l t o n i a n with D = 2.44 c m - t, E / D = 0 and g = 2. T h e d a s h e d line was c a l c u l a t e d a s s u m i n g D=l.75cm t E / D = 1 / 3 and g = 2 .

fitting procedure, 47.1 nmol of the S - - 2 spin state were found for the data of Fig. 3A. Since the volume was 0.150 ml, this represents a sample concentration of 0.314 mM Mn(IIl). The value of gay was set at 2 during these simulations. Varying this parameter did not improve the fits. On the contrary, the fit was noticeably degraded when gay was set to 1.9 for example. The effect of subtracting additional S = 5 / 2 impuri~ frnm the data was investigated - up to a net amount of 5% (relative to total manganese). This did not significantly affect the value of the zero field splitting parameters found from the fitting process. The magnetization difference data (sample minus control) of sodium dithionite reduced M n S O D taken between 1.9 and 200 K at magnetic fields of 1.0, 2.0 and 4.0 tesla are presented in Fig. 4. "~ This nested family of curves can be fit assuming a spin S = 5 / 2 ground state, but cannot be fit assuming any other spin state. 2 Therefore these saturation magnetization data confirm Mn(ll) as the oxidation state of the metal center in reduced MnSOD. An axial (E/D = 0.027) spin S = 5 / 2 Hamiltonian was assumed in the fitting to the data shown in Fig. 4. The value for E/D was derived from the E P R spectrum [9]. The best fits to the data were found with D = +0.50(5) cm - t and 53.1 nmol of S = 5 / 2 (after subtracting the 2% impurity signal - now assumed to be S = 2 ferrous species). As the volume was 0.160 ml, this represents a sample concentration of 0.332 mM Mn(II). In all, we recorded magnetization data on three native and two reduced samples. The data shown in Figs. 3 and 4 represent our most reliable results in

2 Past susceptibility studies have typically been performed at a single, low magnetic field, often at relatively high temperatures. In such cases two measurements are required to determine the spin: the Curie law stope (the slope of versus l / T at high temperatures) and the metal content (by independent analysis) of the sample. Combining these separate measurements gave the spin per metal ion. In the present context and contrary to previously held beliefs [12] it is now not necessary to perform a separate measurement to determine the concentration of the paramagnetic species being studied in order to determine the spin. Rather the spin is determined from the form of the data. Fitting the entire data set of four fixed-field saturation magnetization curves using simulated magnetization curves calculated without approximation from the spin Hamiltonian determines: (I) the spin, (2) spin concentration. (3) zero field splinings and (4) average g value for the sample. For this procedure to work it is crucial, as was the case in previous susceptibility studies, to prepare the sample in a single redox state with a minimum of impurities and to quantitate the impurities that are present. 3 Data were also recorded in the same temperature range at 0.5 tesla. This field has been omitted from the figure because it is obscured by the data at the higher magnetic fields. The theoretical fit was performed on the complete data set taken at all four magnetic fields.

,.4!

165

---

r

.~

0.6

I

~ ~ 1 . ~

f

-

t.O

0.8

~

....-o-

!2

a

T--

t

i T (K)

~.-.~ _...., q

_~ 300~

0.4

0.2 40

t ..

1 0.2

! 0.4

t 0.6

80

103/T ! 0.8

~20

(103K-I) 1 1.0

~60

,

I 1.2

13 H / ( k T )

Fig. 4. Magnetization data for reduced MnSOD from Thermtts thermophilus, pD 6.4, 1.9-200 K. 4-fold excess of sodium dithionite. A contribution of 2 3 (relative to manganese) of an S = 2 (Brillouin function) impurity has been subtracted from the data. Data are at applied fields of 4.0 (e), 2.0 ( o ) and 1.0 ( - ) tesla. For clarity, about one-third of the data points have been edited from the high temperature region without changing the apparent scatter in the data. The solid lines were calculated from a spin S = 5/2 spin Hamiltonian with D = 0.50 cm - t , E / D = 0.027. g = 2 for 53.1 nmol (0.160 ml). Inset: all of the high temperature data, including that at 0.5 tesla ( zx), have been plotted as susceptibility against inverse temperature. For clarity, the data at each lower field have been offset by an additional 20 n J / ( T z sample). The lines indicate the fit used at each field to determine the intercept which was set to zero in the main figure.

terms of freedom from paramagnetic contaminants (as determined by EPR), overall signal-to-noise and importantly, the fact that both were drawn from the same stock solution of enzyme. It is to be stressed, however, that the spin states and zero field splitting parameters determined for oxidized and reduced M n S O D were in all cases the same (within noise) as those we report. Thc-e is a small discrepancy between the values of 0 3 1 4 mM and 0.332 mM obtained for the spin concentration determination from the data of Figs. 3 and 4, respectively. Based on the scatter about the theoretical fits in the two data sets, at the 00% level of confidence, the error in these quantitations is less than + 3 % , or +0.01 mM. Also, control experiments .using viscous solutions have convinced us that any variability in these results due to error in volumetric delivery of sample to holders is at most _+4%, or +0.013 mM. Consequently, for purposes of comparing the spin concentration determinations, the uncertainty can be considered at worst +0.023 mM and therefore, the difference between the two results (0.332 - 0.314 = 0.018 mM) is not significant.

166 We have previously established the validi:y of our quantitation methods during studies with a number of metalloproteins where it was possible, with a high degree of certainty, to independently determine the concentration of the species of interest spectrophotometrically. The systems to which we refer are sulfite reductase [22], ferredoxin I! [23], uteroferrin and cytochrome c (J. Peterson and E.P. Day, unpublished data). On the basis of more than a dozen such sets of data, we have found that provided the presence of paramagnetic contaminants is low (as determined by E P R ) and provided some independently measured parameters for the spin Hamiltonian to be used are available (e.g. g-values), then the spin quantitation obtained is always reliable. Typically, the disagreement between the spectrophotometrically determined concentration of the paramagnet and that found by fitting the magnetization data is + 8 % , or less. Now, taking the mean average of the present results as the best estimate of the enzymatic manganese concentration in these samples and expressing the error in terms of the uncertainty in the fit at the 99% level of confidence ( + 3 % ) plus the maximum reasonable uncertainty in sample volume ( + 4%), we obtain 0.32(2) m M to two significant figures. This error estimate of +0.02 mM ( + 7%) compares very favorably with uncertainties in measuring the manganese content of metalloproteins by more conventional methods (e.g., 24) and is in keeping with the findings of the cumulative magnetization studies noted immediately above. The fits to the data of Figs. 3 and 4, in conjunction with E P P measurements, unambiguously determine the ground state parameters for the manganese in both common oxidation states of Thermus thermophilus MnSOD. With this knowledge, it is possible to fit a family of saturation magnetization curves collected for a sample of unknown redox composition to determine the mixture of oxidation states (or confirm the presence of only one). This was done for a sample of the native M n S O D after first measuring its absorption spectrum. The amount of adventitious S = 5 / 2 impurities was checked (and found to be low) by a parallel E P R measurement. Using this procedure, the extinction of the visible region absorption maximum was found to be • 4s0 = 950 (80) M-= cm-1 (per manganese). The sample was subsequently thawed under argon, diluted volumetrically and used to determine the difference extinction coefficient at 480 nm. Hydrogen peroxide was employed as the reducing agent since our E P R experiments had shown that with this reductant the reaction could be performed aerobically. The value of A~4s0 (native minus reduced) was found to be 740(60) M cm - j (per manganese). These determinations were used to calculate the scales of the ordinate axes in Fig. 1A and B. The uncertainty of +__8.5% in the values of these extinction coefficients reflects the increased noise

levels of these data relative to those of Figs. 3 and 4 (i.e. _+4.5% at the 99% level of confidence for the spin quantitation) plus the possible volumetric error ( + 4% maximum) in loading the magnetization sample holder. Discussion

The properties of some selected microbial M n S O D ' s are listed in Table I. The properties of other well documented (mitochondrial) M n S O D ' s lie within the same range. While the activity per manganese varies by only a factor of two over the examples listed, the extinction coefficient (at 470-480 nm) per m a n g a n e s e varies by a factor of five. It has been noted that both the electronic absorption and C D spectra (where known) of bacterial M n S O D ' s are very similar in all respects except intensity [17]. This may well be due to underestimates of some measured extinction coefficients caused by the presence of reduced enzyme in samples believed to be 100% in the native state. Since the normal catalytic cycle involves alternate reduction and reoxidation of the manganese following successive encounters with superoxide radicals [19] variations in the M n ( I I ) / M n ( l l I ) composition of samples would not be expected to contribute to differences in measured specific activity. T h e present result of r4s0--950 M -~ cm -~ (per manganese) (/t~4s 0 = 740 M - t c m - i (per manganese)) agrees with the values previously found for the Thermu~ thermophilus enzyme, the Thermus aquaticus enzyme and ako, is reasonably consistent with the result reported for the enzyme from Bacillus stearothermophilus (i.e. those M n S O D ' s isolated from thermophilic bacteria); whereas the extinction coefficients (470-480 nm) of the other M n S O D ' s are all lower (Table I). T h e r e are two noteworthy features of these comparative data. Firstly, on a per manganese basis, the magnitude of the extinction coefficient does not seem to depend upon the oligomeric nature (i.e. dimer or tetramer) of the molecule. Secondly however, the magnitude of the extinction coefficient per m a n g a n e s e does a p p e a r to be significantly lower than our present result in those cases where the manganese per m o n o m e r ratio is appreciably greater than 0.5. If this observation is not simply circumstantial, then it may indicate an inequivalence of the metal coordination sites in some MnSOD's, where approximately half the manganese may be preferentially stabilized as Mn(ll) in certain circumstances. The enzyme from Escherichia coli is a case in point. It is not known with certainty if the two manganese binding sites per molecule are equivalent, since there is no crystallographic data concerning this system available. Moreover, the only group to report E P R m e a s u r e m e n t s made on this M n S O D under conditions capable of detecting enzymatically active Mn(ll) (i.e. at liquid helium temperatures) found

167 TABLE I Properties of selected microbial manganese superoxid¢ dismuta~es

Source

Ouaterna~' ~ 34~ Mn per Mn per Specific b structure oligomer molecule monomer activity (units/mgl

Bacillus Stearothermophilus Escherichia coil

dimer

45,487 d 0.90 ~

0.45

dimer

39,500 ~ 1.80 h

0.90

Rhodopseudomonas sp.~eroides Saccharomyces ceret 'isiae Streptococcus m mutans Therraus aquaticus

dimer

37,400 ~ 1.!0 i

0.55

tetramer

92.460 d 3.80 k

0.95

dimer

40,250 ~ 1.85 ,

0.93

tetramer

84,000 '

1.90 ~

0.48

tetramer

83,000 p 2.24 ~

056

Therrtlit$ thermophilus

Activity ' Absorption E.... • ...... per nmol maximum M t cm ~ M -~ cm Mn (nm) per molecule pcr Mn

1,250 63 (pH 7.8. 25°C) 3,800 83 (oH 7.8, 25°C) 2,6tVO ~8 (pH 7.8. 25~C) 3,980 i 97 (pH 8.2. 25 °C) 5.500 120 (pH 7.8, 30 °C) 2,700 t19 (pH 7.5, 23°C) 4,300 ~ 128 : (pH 7.5, 23 ~C)

Refc>:~ce

480

700 f

780

15. 26, 27

473

400

220

3

475

54n

-190

16

480

1 100

290

17

473

560

300

1I

478

2000 o

1050

18

a~O

20a5

910

l

a b c d e f s

Determined by SDS gel electrophoresis. The definition of a unit of specific activity and the method of assay are as descri0ed by M,Lt,:a an Fndovich [25]. Per nmol manganese; that is, specific activity× M, x 10 -6 +number of manganese i~ as per molecule. Determined by amino acid sequence analysis. Determined by neutron activation analysis. Mean value, recalculated using the improved value of Ally, given in the footnote on p. 72 of Ref. 15. Determined by sedimentation equilibrium. Determined calorimetrically following wet ashing. i Determined by gel filtration. i Determined by atomic absorption spectrophotometry. k Determined by X-ray fluorescence spectro~opy. i Assayed according to the method of Marktund and Marklund [28] m Fraction I of Ref. 11. n Determined from the EPR signal intensity of hexaquo Mn(ll) following acid denaturation. o Calculated from Arabs= 0.238 c m - i given in Ref, 18. P Mean value of determinations by gel filtration and sedimentation equilibrium. q The value originally reported was 14000 [1], We have redetermined this to be the stated value. r Calculated from the specific activity (4300 units/rag) of a preparation we determined to contain 2.8 manganese ions per molecule.

their samples to either have, or develop on standing, s i g n i f i c a n t M n ( l l ) c o n t e n t [7]. The zero field splitting of a number of mononuclear M n ( l l l ) c o m p l e x e s in o c t a h e d r a l e n v i r o n m e n t s h a v e b e e n m e a s u r e d in t h e r a n g e - D = 1 to 5 c m - t [29,30]. Although the magnitude of the result for the native M n S O D lies w i t h i n this r a n g e , t h e sign o f t h e z e r o f i e l d s p l i t t i n g f o r t h e e n z y m e is p o s i t i v e . H o w e v e r , t h e geo m e t r y a r o u n d t h e M n ( l l l ) in M n S O D a p p e a r s to b e roughly trigonal bipyramidat, with four protein ligands a n d p e r h a p s o n e s o l v e n t m o l e c u l e in t h e first c o o r d i n a t i o n s p h e r e [2,311. C o n s e q u e n t l y , o c t a h e d r a l M n ( l l l ) c o m p o u n d s w o u l d n o t b e e x p e c t e d to b e a p p r o p r i a t e models for native MnSOD. A somewhat analogous s i t u a t i o n e x i s t s in t h e i n t e r p r e t a t i o n o f t h e d a t a f o r r e d u c e d M n S O D , in t h a t o n l y o c t a h e d r a l l y c o o r d i n a t e d model complexes of Mn(ll) have been studied extensively. In t h i s c a s e h o w e v e r , t h e o b s e r v e d z e r o field s p l i t t i n g o f D = + 0.50 c m - t f o r r e d u c e d M n S O D a n d its E P R s p e c t r u m ( F i g . 2) a r e c o m p a r a b l e to t h o s e f o r a n u m b e r o f o c t a h e d r a l M n ( l l ) s y s t e m s [32,33].

Further interpretation of the metalloprotein data requires studies on structurally well characterized, mononuclear, model complexes of Mn{II) and Mn(lll) in v a r i o u s c o o r d i n a t i o n e n v i r o n m e n t s , T h e r e a r e a number of questions arising from the current work that s u c h m o d e l s t u d i e s m i g h t r e a s o n a b l y b e e x p e c t e d to assist in r e s o l v i n g . F o r i n s t a n c e , is t h e sign o f t h e z e r o f i e l d s p l i t t i n g o b s e r v e d for t h e n a t i v e M n S O D t h e s a m e (i.e. p o s i t i v e ) in all t r i g o n a l b i p y r a m i d a l M n ( l I l ) c o m p l e x e s o r d o e s this d e p e n d u p o n t h e p a r t i c u l a r l i g a n d s ? A l s o , d o e s t h e five c o o r d i n a t e l i g a n d e n v i r o n m e n t s t a b i l i z e M n ( I I I ) r e l a t i v e to M n ( l I ) a n d thus, c o u l d t h e " a u t o r e d u c t i o n ' o b s e r v e d in s o m e s a m p l e s b e associated with increased (or decreased) hydration of t h e m e t a l i o n ? U n f o r t u n a t e l y , this a r e a o f m o d e l l i n g s e e m s to h a v e b e e n l a r g e l y o v e r l o o k e d [34,35] a n d t h e relevant compounds are not presently available. T h u s far, i r o n c o n t a i n i n g s u p e r o x i d e d i s m u t a s e s (FeSOD's) and MnSOD's are apparently indistinguisha b l e in t e r m s o f t h e c r y s t a l l o g r a p h i c a l l y d e t e r m i n e d l i g a n d g e o m e t r y a r o u n d t h e m e t a l i o n s [27]. O n t h e

168 other hand, the axial zero field splitting of both redox states of the manganese in M n S O D from Thermus thermophihts reported here contrasts with the rhombic splitting of the ferric and ferrous forms of F e S O D ' s [36,37]. It is tempting to speculate that substitution of manganese for iron in F e S O D leads to a rhombic Mn(II) which is catalytically inactive towards the superoxide radical. The native FeSOD's which retain activity when substituted with manganese [38-41] are of lower specific activity than most and may represent a comprise situation with an intermediate electronic structure. Investigation of the E P R spectra at temperatures below 20 K of manganese-substituted FeSOD's will test this hypothesis. Finally, it should be noted that this kind of biophysical study involving a S Q U I D magnetometer has until now been concerned mostly with iron containing proteins, where it has been possible to perform M6ssbauer spectroscopic measurements on samples in addition to collecting saturation magnetization data [12,13]. Since the majority of metalloproteins either do not contain iron, or cannot readily be enriched with STFe this previous strategy is not as widely applicable as o n e might wish. Consequently, the success of our present protocol in dealing with M n S O D is significant in that it provides an illustration of how the ground state magnetic properties of metalloprotein derivatives may be directly and quantitatively determined in the most general case. Acknowledgements This work was supported by T h e National Institutes of Health G r a n t GM-32394 (EPD) and University of Alabama Research Grants Committee Project 1521 (JP). References 1 Sato, S. and Nakazawa, K. (197b,) J. Biochem. 83, 1165~1171. 2 Ludwig. M.L., Pattridge, K.A. and Stallings, W.C. (1986) in Manganese in Metabolism and Enzyme Function (Schramm, V.L. and Wedler, F.C., eds.), pp. 405-430, Academic Press, Orlando. 3 Keele, B.B., Jr., McCord, J.M. and Fridovich, 1. (1970) J. Biol. Chem. 245~ 6176-6181. 4 Dingle, R. (1966) Acta Chem. Scand. 20, 33-44. 5 Davies, T.S., Fackler, J.P. and Weeks, M.J. (1968) lnorg. Chem. 7, 1994-2002. 6 Lawrence, GD. and Sawyer, D.T. (1979) Biochemistry 18, 30453050. 7 Fee, J.A., Shapiro, E.R. and Moss, T.H. (1976) J. Biol. Chem. 251, 6157-6159. 8 Villafranca, J.J., Yost, F.J. and Fridovich, 1. (1974) J. Biol. Chem. 249, 3532-3536. 9 Goodin, D.B, (1983) Ph.D. Thesis, University of California, Berkeley, CA. USA.

10 Morgenstern-Badarau, 1., Michelson, M. and Laversanne, R. (1987) Reel. Trav. Chim. Pays-Bas, 106, 243. 11 Vance. P.G., Keele, B.B. Jr and Rajagopala~i, K.V. (1972)J. Biol. Chem. 247, 4782-4786. 12 Day, E.P., Kent, T.A., Lindahl, P.A., Miinck, E., Orme-Johnson, W.H., Roder, H. and Roy, A. (1987) Biophys. J. 52, 837-853. 13 Day, E.P., David, S.S., Peterson, J., Dunham, W.R., Bonvoisin, J.J., Sands, R.H. and Oue, L., Jr. (1988) J. Biol. Chem. 263, 15561-15567. 14 Nelder, J.A. and Mead, R. (1965) Computer J., 7, 308. The computer program was developed by Thomas A, Kent of the Chemistry Department at Carnegie-Mellon University. 15 McAdam, M.E., Fox, R.A., Lavelle, F. and Fielden, E.M. (1977) Biochem. J. 165, 71-79. 16 Lumsden, J., Cammack, R. and Hall, D.O. (1976) Biochim. Biophys. Aeta 438, 380-392. 17 Bjerrum, M.J. (1987) Biochim. Biophys. Acta 915, 225-237. 18 Sato, S. and Harris, J.l. (1977) Eur. J. Biochem. 73, 373-381. 19 Abe, Y. and Okazaki, T. (1987) Arch. Biochem. Biophys. 253, 241-248. 20 Weisiger, R.A. and Fridovieh, 1. (1973) J. Biol. Chem. 248, 3582-3592. 21 McCord, J.M., Boyle, J., Day, E.D., Rizzolo. L.J. and Salin, M.L. (1977) in Superoxide and Superoxide Dismutases (McCord, J.M. and Fridovieh, !., eds.), pp. 129-138. Academic Press, New York, 22 Day, E.P., Peterson, J., Bonvoisin, J.J., Young, L.J., Wilkerson, J.O. and Siegel, L.M., Biochemistry 27, 2126-2132 (1988). 23 Day, E.P., Peterson, J., Bonvoisin, J.J., Moura, I. and Moura, J.J.G., J. Biol. Chem. 263, 3684-3689 (1988). 24 Beyer, W.F., Jr. and Frivovich, 1., Anal. Biochem., 170, 512-519 (1988). 25 McCord, J.M. and Frivovich, I. (1969) J. Biol. Chem. 224, 60496055. 26 Brock, C.J., Harris, J.l. and Sato, S. (1976) J. Mol. Biol. 107, 175-178. 27 Brock, C.J. and Walker, J.E. (1980) Biochemistry 19, 2873-2882. 28 Markiund, S. and Marklund, G. (1974) Eur J. Biochem. 47, 469-474. 29 Kennedy, B.J. and Murray, K.S. (1985) Inorg. Chem. 24, 15521557. 30 Kennedy, B.J. and Murray, K.S. (1985) Inorg. Chem. 24, 15571560. 31 Parker, M.W. and Blake, C.C.F. (1988) J. Mol. Biol. 199, 649-661. 32 Dowsing, R.D., Gibson, J.F., Goodgame, M. and Hayward, P.J. (1969) J. Chem. Soc. Ser. A, 187-193. 33 Yonetani, T., Drott, H.R., Leigh, J.S., Jr., Waterman, G.H. and Asakura, T. (1970) J. Biol. Chem. 245, 2998-3003. 34 Wieghardt, K. (1989) Angew. Chem. Int. Ed. Engl. 28, 1153-1172. 35 Vincent, J.B. and Cristou, G. (1989) Adv. lnorg. Chem. 33, 197-257. 36 Slykhousc, T.O. and Fee, J.A. (1976) J. Biol. Chem. 251. 54725477. 37 Whittaker, J.W. and Solomon, E.I. (1988) J. Amer. Chem. Soc. 110, 5329-5339. 38 Gregory, E.M. and DaFper, C.H. (1983) Arch. Biochem. Biophys. 220, 293-300. 39 Meier, B., Barra, D., Bossa, F., Calabrese, L and Rotilio, G. (I982) J. Biol. Chem. 257, 13977-13980. 4~1 Pennington, C.D. and Gregory, E.M. (1986) J.Bacter. 166, 528332. 41 Martin, M.E., Byers, i$.R., Olson, M.O.L, Salin, M.L., Arceneaux. J.E.L. and Tolbert, C. (1986) J. Biol. Chem. 261, 93619367.