J. Mol. Biol. (1991) 221, 1325-1344

Structure of NADH Peroxidase frFm Streptococcus faecalis 1OCl Refined at 246 A Resolution T. Stehle’, S. A. Ahmed2t, ‘Institut

A. Claiborne’

and G. E. Schulz’

fiir Organische Chemie und Biochemie der Universittit Albertstrasse 21, D-7800 Freiburg i.Br., Germany

‘Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103, U.S.A. Received 20 March 1991; accepted 14 June 1991 The crystal structure of NADH peroxidase (EC 1.11 .l. 1) from Streptococcusfaecalis lOC1 (Enterococcus faecalis) has been refined to a resolution of 2.16 A using the simulated annealing method. The final crystallographic R-factor is 17.7 o/o for all data in the resolution range 7 to 2.16 8. The standard deviations are PO15 A in bond lengths and 30” in bond angles for the final model, which includes all 447 amino acid residues, one FAD and 369 water molecules. The enzyme is a symmetrical tetramer with point group D,; the symmetry is crystallographic. The redox center of the enzyme consists of FAD and a cysteine (Cys42), which forms a sulfenic acid (Cys-SOH) in its oxidized state. A histidine (HislO) close to Cys42 is likely to act as an active-site base. In the analyzed crystal, the enzyme was in a non-native oxidation state with Cys42 oxidized to a sulfonic acid Cys-SO,H. The chain fold of NADH peroxidase is similar to those of disulfide oxidoreductases. A comparison with glutathione reductase, a representative of this enzyme family, is given.

Keywords: NADH peroxidase; flavoenzymes; X-ray structure; cysteic acid; Streptococcus faeculis lOC1

1. Introduction The NADH peroxidase (NPXase$) from Streptococcusfaecalis (Dolin, 1975, 1982; Poole & Claiborne, 1986; Claiborne et al., 1991) and the alkyl hydroperoxide reductase from Salmonella typhimurium (Jacobson et al., 1989; Tartaglia et al., 1990) represent the two known flavin-dependent hydroperoxidases, or “peroxide reducfases”. The unusual flavinlinked functions of the two enzymes, which involve heterolytic scissions of the -O-O- bonds of the respective peroxide substrates, are chemically similar to those of the well-characterized flavoprotein disulfide reductases such as glutathione 7 Present address: Laboratory of Biomedical Pharmacology, NIDDK, NH. Bethesda, Maryland 20892, U.S.A. 1 Abbreviations used: NPXase, NADH peroxidase from Streptococcus j’uecalis 1OCl; GRase, glutathione reductase; Cys-SOH. cysteine-sulfenic acid; EH,, reduced NADH peroxidase; m.i.r.. multiple isomorphous replacement; Q, standard deviation; r.m.s., root-mean-square. (K)42%2836/91/20132.5-20

$03.00/O

reductase (GRase: Williams, 1976; Thieme et al., 1981). Instead of a redox-active disulfide group, the streptococcal NADH peroxidase utilizes a unique stabilized cysteine-sulfenic acid (Cys-SOH; Poole & Claiborne, 19896) in the catalytic reduction of H,O,. This second redox center alternates between sulfenic acid and thiol redox states during turnover. Reduction of the enzyme with one equivalent of NADH yields a two-electron reduced species (EH,) which is spectroscopically very similar to the EH2 form of GRase (Poole & Claiborne, 1986). N-terminal sequence comparisons between NPXase and GRase from Escherichia coli yielded alignments of sequence fingerprints for FAD-binding and of the cysteine residues (Poole & charge-transfer Claiborne, 1989a). An overall alignment resulted in amino acid residues (Ross & 21% identical Claiborne, 1991). The high-resolution structure of NPXase should contribute to three lines of research. (1) The combination of flavin and Cys-SOH redox centers represents

1325

a novel

aspect

for 0

enzyme 199l

Academic

catalysis.

Of

Press Limited

1326

T. St&e et al

particular interest is the stabilization of the sulfenic acid in the enzyme, since such derivatives generally have only transient existence in solution. (2) With the cloned and overexpressed gene in hand (Ross & Claiborne, 1991), mutant’s can be produced according to the three-dimensional structure, which will allow for a detailed comparison between NPXase and the related NASH oxidase (Ahmed $ Claiborne, 1989aJ) with respect to reaction mechanism and substrate discrimination. (3) Since the Gram-positive streptococci have diverged from the Gram-negative bacteria for over one billion years (Priebe et al., 198&S),a comparison between NPXase and t’he various flavin-dependent disulfide reduct’ases such as GRase (Karplus & Schulz, 1987), lipoamide dehydrogenase (Schierbeek rt al.. 1989). thioredoxin reductase (Kuriyan et al., 19x9) and mercuric reductase (Moore et al., 1989) should be of evolutionary interest (Pet,sko. 1991). Recently, we reported the 3.3 Lh (1 !I =&I nm) st’ructure of NPXase (Stehle et nl.. 1990), which established that the domain st,ructure and the monomer chain fold are similar to that of GRase. t)he structure of which initialized the detailed studies of this family of flavoproteins. Now, we report the NPXase structure refined to a resolution of 2.16 *‘I. 2. Materials

and Methods

NADH peroxidase was purified from A’. J~~calis IN I (ATCC 11700) by a procedure based on that, of Poole & (‘laibomr (1986). A4pprox. l8Og of frozen cell paste was suspended, broken and carried through the ammonium sulfate fractionation as described for the streptoc*oc*cLal 1989~). Tht X.4DH oxidase (Ahmed B (‘laiborne. 2.26 M-ammonium sulfate suprrnatant \vas applied to a

DE,52 column (60 c-mX 5 cm) equilibrated in 50 m-Mphosphate (pH 6%), containing 2.26 &I-ammonium sulfatci and OB~WEDTA. The rolumn was washed with I I of 6.34 M-ammonium sulfate in the same buffer prior to starting a 3 1 gradient from 2.34 M to 1.39 M-ammonium sulfate. A final column wash with I.5 I I.39 M-ammonium sulfate eluted the enzyme. which was pooled and dialyzed for the next column step. The dialyzed protein sample was applied to a 2nd DE52 column (30 cm x 6 cm) equilibrated in the same WM-Na(?l containing buffer (Poole & C’laiborne. 1986). washed with 700 ml of t,his buffer. then eluted with a I.5 I gradient from 0.2 t,o 0.4 M-NaU. The pooled enzyme was caonc-entrat)ed to a final volume of less than 15 ml. applied to a Sephacrvl S-200 column (IOOcm x Scam). and elu&ed in phosphate buffer (pH 6.8). Fractions of specific activity greater than about, 45 units, mg (using EZSO[l ‘+0] = 10 to determine protein concentrations) werr combined and dial-vzed for h$rox~lapatite chromatography. The enzyme was applied to a Bio-Gel HTP column (20 cm x 2.5 cm) equilibrated in 10 mnlphosphate (pH 7.0). then washed with 200 ml of the buffer before elution with a I I gradient from OGI M to 0.2 RIphosphate. Fractions of I50 to 160 units/mg specifit activity (using edSO= 10,900 M-’ c&n-’ for the bound flavin for determining the protein concentration) with A,,,/A,,o ratios G8.1 were pooled, concent,rated to 1.8 mg/ml. dialyzed against, 50 rnM-potassium approx

phosphat,e at pH i.0 JJ~US 0% rnw-EDT;\. anti t’rozerl ill portions at - %O’(‘. The scaled-up protoc.ol givrs about 30 mg of pure protein from -CHI of bacstrrial (.ulturta. ‘1‘11~~ overall yield is 30°q,. The ac,tivit~-to-favin ratio (AFK,. i.e. thr ratio of units in the standard asna~- to enzvnlca absorbance at WI nm) is about 700 as drrivrd f&n, ac$ivitJ. of I ,iT, :W,= 50.300 for a monomer and a spfvitic urlits/mg. J’l, was c~al(~ulatetl from the st~quener (Ross k Claiborne. 1991) JJ~LIS FAI). (‘r:\stalx were grown it] sitting drops by vapor tlitfurio~~ at 20 to Ib’(’ using a prot,rin stock of IO mg,/ml in 50 ml

potassium phosphate at pH 7.0 with 0.5 nr~-El)T.~I and 2 m;ll-dithiobhreitol (Stehle rl ~1.. 1990). The rcAser\oir buffer contained. in addition, 2.05 n-amntoniunl sult’atcL (Schwarz-Mann l%rapurr). SPM-FAD and 3 msSaN,. For t,hr crgstallization setups. 20 &droplrt~s MWC prepared bg mixing (with a Gilson l’ipe‘tmalr) 10~1 01 protein solution with IO ~1 rrsrrvoir buffer. Four of thesca droplets were set on I small Petri dish (35 mm x IO nbn1: Kunc. inc.). Reservoir buffer (2.5 ml) \ViIS added ttr e1~f.h of 2 other small I’rt,ri dishes. Thd \\.it II absorbent tissue. This shipping t)rotc,c*c)l ha(l II,) ad\~c~rsr~ erect on reflec+tion profiles or diffrac+iorr po)vC’r It, shoultl be mrntionrd. however. that t,htx c.rystals USA/ for S-IX> analysis wert’ at least 2 months old. Dissolved c,rvstals ot an age of I year sho\Vrd less than I ‘I,, it(.tivit,\- rc~tnaining LL’r may t hrrrforc, c~onc*lude that tlirb ~~r~stxlliric~ (~nz~.mc~ had aged prior to. ot‘ ciuring, N-ray analysis alItI that the described struc+urr rrpresent: CI an ynzyrnci that is nor)active to a high prrc~entagt*. Ilost likely t trc, ICI+ r)t activity is causrd bv the oxidation of th(L rc&s-ac~tcv4~ sulfenip acaitl to a sulfonic* acid in the cr?.stal as &tr~c+rtl iti the stru(‘ture analysis: inac~tivatiorr of KPNasr by H2( )I has also been shown to lead to the sulfonic acsid ~lerivativ~~ (Poole & C’laiborne. I !389h).

The Kl’Sase car@als belong to space group li’22 u 11h unit cc.11dimensions (I = 77.2 I4. h = iS4.5 A anti ,’ = I l5.9 x. They cont,ain I monomer (X, =:50.30O):as?-rnntetri~ unit. At ii7 y,, tht> solvent c.ontent is vet’>- high. The carvstals diffract to 2.1 d resolution. 0>3tals of t,his c~nzyrne iti thr same spaqta group with slightly different wII dimr~nsions and 2 rnonornrrs:aspmr,lrtric~ unlit have been rt,portcJd t,J. Sohiering I+ n,Z. (1989). In order to improve the initial nlultiph~ 1son1ort)ho(ls replacement (rn.i.r.) model of the XPXasr structurrh al 9-3 A resolut,ion (Stehlcl I,! trl.. 199(j), a neM’ rrati\r data sit was c~ollrc*ted to I.16 X resolution using s~nchrot ran radiation on heam-line S I1 of t,hr EMRL outstatioll at I)ESY (Hamburg). The data aerc~ c.ollec*trd on an image plate from 1 cryst,al using a wavelength of 0.96,.$. which was c*hosrn so as to minimize radiation damage as well as absorption effec+. High-resolution dat,a wert’ c,ollectrd b>

Structure of NADH

The refinement protocol is given in Table 2. In the first round, native diffractometer data between 10 and 3.3 A resolution were used to refine the initial n1.i.r. model of Stehle et al. (1990). All B-factors were set t’o 180A’ as derived from a Wilson plot (data not shown). The initial and the resulting &factors in this resolution range were 48.6% and 25,0°‘,c, respectively. All subsequent rounds of refinement were based on the merged native data set (Table 1). They were carried out at higher resolution, first in range 10 to 2.16 A and later in range 7 to 2.16 A (Table 3). After the 2nd and all subsequent rounds the model was inspected using (2F, - F,)exp(ia,) and (F’, - F,)exp(ia,) electron densitv maps. Mispositioned residues and incorrect conformations were changed manuallv using the program FRODO (Jones, 197X) on interactive graphics (model PS330, Evans & Sutherland. L’.S.A.). When the 2nd round was completed. the newly calculated (2F, -F,)exp(icc,) and (F, - F,)exp(ia,) maps showed clearly the density for 17 additional C-terminal amino acid residues. These had not been included in the m.i.r. model (Stehle et al., 1990) dur to incomplete sequence data at that time. Moreover. at this stage the 1st oxygen atom at the active-site Cys42 residue was detected. Aft.er the 3rd refinement round, the highest peaks in the (F,-F,)exp(ia,) map resulted primarily from side-chain errors, solvent molecules. and a few misplaced carbonyl oxygen atoms. In the regions of residues 28 to 31 and 238 to 242 the chain was found to be out of register and had to be shifted by 1 residue. From round 4 on: water molecules were incorporated at places where the density manually in the (F,-F,)exp(i~,) map was at least 40. Before including them, they were inspected visually t.o make sure that they fulfil the geometric requirements for hydrogen bond formation. The initial B-factors of the water molecules were always set to 25 8’. On completion of the 5th round, the (E’,-F,)exp(ia,) map showed 2 very high distinct peaks at Cps42. which were interpret)ed a.s 2nd and 3rd oxygen atoms. converting this cysteine to a sulfonic acid after it had already been changed to a sulfenic acid in

Table 1 Data collection statistics Diffractometer m-3.3 11.860 98.2

Resolution (A) Reflections (Jompleteness (?h)

Rsyma(46) kb (5%)

Synchrotron/ image plate

Merged

262.16 34,899

00-2-16 37,857

83.8 I@6

92.0

4.3

1325

Peroxidase

61

"R,,, is defined as ~i,h~r(ll(i,hkl)-(I(hkl))(/Z,,h,,(l(hkl)), where i runs through symmetrically related reflections. bR, is defined as 2 x ZlFl -PZljZ(J’l fF2). For diffractometer data (modified model PZ,, Nicolet-Siemens), Fi and F2 are the st,rurture factor amplitudes of symmetry-related reflection zones h/cl and hki. For merging, Fl and F2 are the structure factor amplitudes of all equivalent reflections in the 2 data sets.

rotatmg 62” in 1” intervals. The data were reduced using the SCRAP and the MOSFLM/IMAGES programs of the EMBL outstation. Without any cutoffs? 57 of these 62 frames were merged together, yielding a native data set containing 34,969 unique reflections out of 67.211 recorded reflections with an R,,, of l@6o/o. The intensities were then converted to structure factors using the program TRCh’CATE (French & Wilson, 1978), which deleted 70 reflections with structure factors below zero. The remaining 34,899 reflections were then merged with the previously collected native 3.3 A diffractometer data set. yielding a, 92% complete native data set with 37,857 unique reflections in t.he range co to 2.16 A (Table 1). In the range 2.20 to 2.16 A the data are still 7796 complete. (c) Course of rejinement Refinement of t’he KPXase structure was carried out with a total of 9 rounds of the simulated annealing method using the program XPLOR (Briinger et al., 1987).

Table 2 XPLOR REPEL’

Round WA” (Mcal/mol) 143 280 27s

27s Pi.5 2i5 275 350 350

WPb (Mcal/mol rad’)

23 0 0 0 0 0 0 0 0

refinement MIX-ld

prntocol

(cycles)

20 15 20

300K’

20OOK’

MIN-2s

50 50

10

.50

25

50 50

-

50

-

SO

1.0

MIN.2

(cycles)

(PS) 106

RREF”

@76 045 til

150 100 50 138 130

100 70 100 100

10 10 IO 10 10

30 30 311

10

30

10 10

30 50

30 30

“WA, weight for the effective energy term accounting for the diffraction data, E, (XRAY) bWP, weight for the effective energy term accounting for the phase information, E, (XRAY). These weights relate the X-ray effective energy terms I%‘, (XRAY) and E, (XRAY) to the empirical potential energy (1 cal=4184J). ‘REPEL, conjugate gradient minimization with soft repulsive potential. tolerance AF =O+S A AF is a limit. If any atom movement exceeds AF, the 1st derivatives of the effective energy krm [EA (XRAY)+Er (XRAY)] are recalculated. dMIN-l, conjugate gradient minimization with CHARMM non-bonded potential, AF =00.5 P\. ‘2OOOK. molecular dynamics at a “temperature” of 2000 K, timestep= 1 fs, AF =@3 A. ‘3OOK, molecular dynamics at 300 K, timestep= 1 fs, AF =62 A. sMIN-2. conjugate gradient minimization, AF =0605 A. ‘RREF, Individual B-factor refinement with standard deviations between B-factors of bonded at,oms and R-factors of atoms connected by an angle restrained to 1.5 A’ and 26 Aa, respertively

1328

7’. Stehle et al.

---.

Table 3 Course of rejinement

using

simulated

trrt,nealGLy Round

Resolution range (A] R-factor (76) Number of reflections Number of amino arids” Number of water molecules r.m.s. deviations of Bond-lengths (A) Bond-angles (deg.) Impropers (deg.) Dihedrsls (deg.) “Residue Cvs42 was modeled as a cvsteine-sulfenic acid in round 3 and as a cysteiv acid in round 5 and the following i i round 8. Gly54 was changed to Asp54, correcting a reading error

round 3. The geometric parameters fbr the cgsteic acid were taken from Hendrickson & Karle (1971). Furthermore, a water molecule (Wat474) was found at an unusual position right on the dyad between the INTERFACE domains of 2 NPXase monomers. In round 7 all solvent molecules were scrutinized critically. leading to a major rearrangement. All temperature factors of the water molecules were again set to 25 ip’. The refinement was halted when the R-factor reached 17.7?& and the corresponding difference Fourier map showed no more interpretable features. The final model of a RiPXase monomer contains 3493 non-hydrogen protein atoms, 53 FAD atoms, and 369 water molecules. The final root-mean-square (r.m.s.) bond length and bond angle deviations are 0.015.!1 and 3.0”. respectively. Details of the refinement are given in Table 3. The refinement’ and all other calculations were carried out on a MicroVAX-I1 and a VAXstation-3100 (Digital Equipment Corp.. [T.S.A.). The central processing unit time for 1 refinement round (see Table 3) was between 3 and 5 days on the lat,ter

machine.

3. Results and Discussion (a) Quality

and accuracy

r~,und?;. Afttar,

m.i.r. model at 3.3A resolution (Stehle et (~1.. 1990) the r.m.s. shift of the main chain atoms is 1.1 ,r\. Furt,her changes are as follows. (1) The refined model now contains all 447 amino acid residues instead of 430. The additional 17 residues form helix ctll at the C terminus. Due to sequence uncertairlties this helix was nob assigned in the 3.3 A st)ructure, although it had density at the 1~ level. (2) The cysteinyl residue at position 42 is now modeled as a cysteic acid Cys-SO,H. (3) Amino acid residues 28 to 31 and 238 to 242 have been shifted by once residue, shortening and lengthening the respective connecting loops. (4) A total of 369 solvent moltscules are now included. (b) C’hain conformation Although the main-chain dihedral angles were not rest,rained during refinement. all but two nor)glycine residues are found in favorable regions of t.hcRamachandran (4.$)-plot,. As can be visualized in Figure 4(a) the main-chain torsion angles for Lysl23

of the model

According to Luzzati (1952), an upper limit for the positional errors of the final model can be determined by plotting the R-factor as a function of resolution. In Figure 1 this plot shows an upper error limit of 023 A. As a more qualitative description of model accuracy we present a part of the final (2F,-F,)exp(ia,) map in Figure 2. This shows the model of the isoalloxazine moiety of FAD together with the corresponding density. All atoms of NPXase are well-defined in their densities with the exception of the side-chains of residues Asp25, Cys(-SO,H)42, Lys51, Asp124 and Arg447. The weak density between Cys42-CA and -CR is possibly due to a mathematical density calculation problem around the dense sulfonic acid (see below). The other four residues are located at the protein surface and are presumably very mobile. A C” backbone drawing of the refined NPXase struct,ure is given in Figure 3. As compared to the

l/f

,/ I 0.3

--.-/-0

0.1

o-2

I o-4

i--0.5

I -.I O-6

2 sin R/X

Figure NPXase.

1. Luzzati

(1952) plot of the retined

The R-factor

has been calculated

from

model oi’ all non-

centric reflections. The lines emerging from the origin arp the theoretical upper limits for co-ordinate errors. tht, lower one is tb2OA and the upper one is 0% P\. The resolution

is given

at. the t,op.

Structure of NADH

Peroxidase

1329

Figure 2. Final (SF,--P,)exp(icr,) map, in stereo, for the isoalloxazine moiety of FAD together with the refined model. The depicted cut level is lo. The strong hydrogen bond between O-&F and Wat490 (see Table 8) accounts for the density protruding from this atom.

(a)

(b) Fig. 3.

T. St&e et al

1330

246

246

Cd)

(e) Figure 3. Stereo drawings of the (I” backbone chain fold of a NPXase subunit. same molecule orientation. (a) Chain fold of a whole subunit together with FAD. given, the vertical axis corresponds to the molecular dyad of dimeric GRase. FAD. (c) NAD-binding domain and 1 dyad together with FAD as a reference. FAD. (e) INTERFACE domain with 1 dyad. The domain names and limits are

All parts of the Figure correspond to t,hcs Th e molecular dyads of the tetramer are (b) FAD-binding domain tog&her with (d) CENTRAL domain together wit,h taken from GRase (see Fig. 9 and text)

Structure of NADH

Peroxidase

1331

Table 4 Assignment qf secondary structures Domain

Residues 1-7 S23 27-32 4349

62-69

FAD-binding

a-Helix

/Z-Strand

81

al a2 a3

72-74 7683 87-94 98-104

lO(i-110 127-129 133-144

lNk156 158-171 174-179

NADbinding

9

190-203 207-209 216218

(a)

r4 a5 DlO a6

224-228 231-235 237-241

(‘EIYTRAL

27.5479 298-312

a7

327-331 334-339 342-349

a8

354-362

I?U’TERFACF I1

0

0

n

90 c

I

I

90

180

40-410 413419 4:13-446

I

Average qY (deg.) Average I) (deg.) Average S. .O (A)

n 270

371-379 388-393 U9

X10 rll -65

(9)

-39 (9) 3a7 (02)

-121 (20) 133 (24) 2.93 (0.1)

360

X, (b)

Figure 4. Chain conformation

of NPXase. (a) Scatter plot of main-chain torsion angles 4 and II/ of the 416 nonglycine residues. There are 11 residues in the left-handed cc-helix region: Asn206 and Lys411 are at C-terminal ends of helices: Ser38, Asn76, Asn127, Leul85 and Asp275 are at (i-t 2)-positions of distorted type-II turns; His87, Met269. Asp291 and Thr384 occur at (i+3)-positions of distorted type-1 turns. The 2 residues found in forbidden regions are Phe332 at (50”, -120”) and Lys123 at (-I?, -50”). (b) Scatter plot of the side-chain torsion angles (x1, xz) of the 35 leucine (A) and 28 isoleucine (0) residues. The residues cluster around the staggered conformations.

and Phe332 are in forbidden regions. Both residues, however, have well-defined densities. Lys123 participates in two reverse turns in the chain segment connecting FAD- and NADH-binding domains: in a type-11 turn (residues 120 to 123, where residue 122 is glycine) and in a distorted type-T t,urn (residues 122 to 125). Phe332 occupies position i+ 1 of a type-II’ turn (Venkatachalam, 1968), where glycine is usually required. The torsion angles of 11 non-glycine residues fall in the lefthanded cc-helical region around (60”, 40”). They are

“In all averages,

the first and the last residues have been

omitted, the r.m.s. deviations are given in parentheses. given in Figure 4(a), showing a marginal preference for Asx (Matthews, 1977). Figure 4(b) shows a scatter plot of the x1 and x2 torsion angles for leucine and isoleucine. For the most part these angles are within the regions for staggered conformation, corroborating the quality of the final model. The observed frequencies of the nine staggered conformations are in good agreement with the statistics of Janin et al. (1978) and the observations of Karplus & Schulz (1987).

(c) Secondary structural rlewt,entx The assignment of secondary structural elements depends on the hydrogen bond criteria applied. For resolution protein structures, Baker & high Hubbard (1984) have shown that peptide hydrogen bonds are best defined by a donor-acceptor distance of less than 3.5 a and an N-H. .O angle of more than 120”. On the basis of these criteria, we have assigned cc-helices and p-strands manually by inspecting hydrogen bonds and torsion angles as calculated in XPLOR. The results are given in Table 4. The overall domain structure and the chain fold

1332

T. Stehle et al.

Figure 5. The P-sheets of NPXase. The lengths of all hydrogen bonds are given as I\;. . .O distance in ,r\

of NPXase resemble those of GRase (see Fig. 9). Accordingly, we assign the FAD-binding, NADbinding, CENTRAL, and INTERFACE domains as in GRase (Thieme et al., 1981). Note that NPXase binds NAD and not NADP as GRase does. The FAD-binding domain consists of a four-stranded parallel /?-sheet (fll$Z,fi3&?7) connected by or-helices (al,cr2,a3) placed below the plane of the paper in Figure 5 and a p-meander (/?4$5$6) located above the plane. The secondary structure of the NADbinding domain resembles that of the FAD-binding domain, except that (1) it lacks an equivalent of the chain extension containing helix ~3, (2) has a preceding fifth strand (BS) in its parallel p-sheet, and (3) has a preceding helix ~4. The CENTRAL domain consists mostly of irregular structures. There is one a-helix (~17) and one b-strand (BlS). As shown in Figure 5, the single p-strand adds a fifth strand to the parallel /?-sheet of the FAD-binding domain at the equivalent sheet edge as the preceding fifth strand BS in the NADbinding domain. Domain INTERFACE consists of a five-stranded antiparallel b-sheet shown in Figure 5, a short helix connecting two of the strands, and three cc-helices at the C terminus. The chain folds of the four domains of NPXase are

shown separately in Figure 3. In total, NPXase contains 11 cr-helices (comprising 28% of the residues) and 21 p-strands (27%) assembled in five sheets. The a-helices are well defined and show the usual average (4,$)-values (Table 4). All helical hydrogen bonds obey the applied criteria, with the exception of Glu192-0.. .Va1196-N in helix a6 with a distance of 3.99.& Here, Glu192-0 is hydrogen-bonded to Wat599 (distance = 2.92 A) and the general course of the helix is not affected. The interactions within the five j-sheets are detailed in Figure 5. The average (4,#)-values in these sheets are in the usual range (Table 4). The hydrogen bonding patterns in NPXase are regular: except for strand 84 of the b-meander in the FADbinding domain. Here, the additional residue Se&O introduces a /?-bulge, while Glu79-0 forms a hydrogen bond to N-60! of the adenine moiety of FAD. When compared with GRase, the B-meander of the FAD-binding domain of NPXase is larger than that of the NAD-binding domain, whereas it is the other way around in GRase. As commonly observed, the hydrogen bonds within p-sheets are somewhat shorter than in a-helices (Table 4), indicating a higher stability of the sheets.

Structure of NADH

Table 5 Salt bridges in NADH Residue 1

Residue 2

LysZ-NZ Glul4-OEl Glul4-OE2 Glul4-OE2 Glul7-OEl Glu49-OEl ArglO6NE LysloG-NZ Argl32-NH2 Argl32-SH2 Glul66-OE2 Asp196OD2 LyslSl-NZ Glul92-OEl Arg216-NH2 Lys225-NZ Asp266ODl Lys351-NZ Lys354-NZ Glu366OEl

Glul02-OEl Arg303-NH1 Arg307-NE Arg307-NH 1 Arg69-NH1 Lysl70-NZ Glul02-OE2 Asp275-OD2 Asp281-ODl FAD-OAl Lysl’lO-NZ Lys376-NZ Aspl95-ODl Lys376-NZ Glu218-OE2 Asp234-ODl Arg270-NH2 Asp386OD2 Glu446-OEl Lys439NZ

peroxidase

Distance (4 325 2.85b 2.80b 2.77b 2.74 3.44 3.16 2.66 3.48 2.82 3.02 2.69 2.79 296 296 3.23 281 2.78 286 366

Domains” l-l l-3 l-3 l-3 l-l l-2 l-l l-3 2-3 2%Prosthetic group 2-2 24 2-2 24 2-2 2-2 3-3 44 44 44

“Domains 1, 2, 3 and 4 are the FAD-binding, the NADbinding, the CENTRAL and the INTERFACE domains, respectively. bThese salt bridges make up the zipper between 2 domains discussed in the text.

Peroxidase

1333

to 124, 219 to 222 and 444 to 447 at the C terminus there are four regions comprising 14 residues with B-factors above 35A2. In Figure 6, a comparison between the B-factors given in (a) and the solvent accessibilities given in (b) shows a good correlation; solvent-exposed chain segments have large B-factors and vice versa, the correlation coefficient being 679. It should be noted that the solvent accessibilities of Figure 6(b) have been averaged over a sliding window of five residues; the correlation coefficient drops to 644 if the accessibilities are taken per residue. When calculating the average main-chain B-factors for each domain, one finds 189 A2, 188 A’, 159 A2 and 138 A2 for the four domains along the chain. The INTERFACE domain is the most rigid one, which is consistent with its large buried surface resulting from the tight intersubunit contact (see Table 6). The prosthetic group FAD is bound tightly in a region of low B-factors. A closer look at its B-factors shows a gradient extending along the dinucleotide as in GRase. The average B-factors of isoalloxazine, ribitol, pyrophosphate, ribose and adenine are 62A2, 61 A’, 63 A2, 1@5A2 and 192 A2, respectively. (f) Solvent structure

(d) Ionic interactions Since the contribution of salt bridges to protein stabilization can be appreciable, we report in Table 5 the 20 salt bridges found in NPXase. An interesting feature involving some of these residues is a zipper-like accumulation of alternatingly charged residues extending from Arg303 (close to the active site base HislO), via Glu14, Arg307, water molecule Wat515 and Glu18 to the surface. As these residues are alternatingly contributed from the FAD-binding and the CENTRAL domain, this system seems to be designed to zip these domains tightly together. A second, somewhat smaller “domain zipper” may be recognized in the interactions of Asp190-Lys376-Glu192 between the NAD-binding and INTERFACE domains. Apart from the above-mentioned residues, we find most of the other 15 salt bridges within one domain, only three are formed between different domains. (e) Chain mobility The average isotropic temperature factor of the refined NPXase structure is 19.5 A* for all nonhydrogen atoms including water, which is only slightly higher than the value of 18 A2 originally taken from a Wilson (1949) plot (data not shown). The average B-factor of the main-chain atoms is as low as 17 A’. In conjunction with the high solvent content of 67%, which is at the upper limit observed for protein crystals (Matthews, 1977), the low B-factor indicated that NPXase has an intrinsically rigid structure. Figure 6(a) gives a B-factor plot along the main-chain. At positions 96 to 99, 123

The NPXase model contains 369 water molecules, all of which have B-factors below 55 A2 and densities above lt~ in the final (2F,-F,)exp(ia,) map. After the refinement, the water molecules have been renumbered according to their final map density starting at number 449 (after the 447 amino acid residues and FAD). The solvent distribution as a function of density is given in Figure 7. Wat449 has the highest observed density at 550; the average is around 20. The average B-factor as a function of density is also given in Figure 7. As to be expected, B-factor and density are correlated negatively. Following the suggestions of Blevins & Tulinsky (1985) and Karplus & Schulz (1987), we consider 56 of these water molecules as an integral part of the enzyme because they have densities above 30 and B-factors below 25 A2. Among these, 25 are buried in the interior of the protein with solvent accessibility zero. Most of these integral water molecules are located in the vicinity of FAD and at the large contact between the INTERFACE domains. This contact accommodates also Wat474 sitting directly on a crystallographic dyad. Wat474 was inserted at a fixed position and was given a B-factor of 20A2; for technical reasons it was not refined with XPLOR. The average B-factor for all water molecules is 33 A’. A total of 328 of the water molecules are within a distance of 3.7 A from the nearest protein atom and belong to the inner hydration shell. The remaining 41 molecules are in a second shell in the range 3.7 to 50 A from the nearest protein atom, where they are fixed by solvent-solvent interactions. Their average B-factor is 40 A2, exceeding by far the overall average.

T. Stehle et al.

1334

50

CENTRAL

NAD-binding

FAD-binding

100

150

200 Residue

250

INTERFACE

300

350

400

450

300

350

400

450

number

J 0

50

100

150

200

250

Residue

number

Figure 6. Mobility and solvent accessibilit? in. R;I’Xase. Residues :3 through 445 are plot,trd. (a) Maill-chain N-f’avtors averaged on a per-residue basis. The domams are indicated at t,he top (b) Solvent. actzxsihilit.~ in ?r’F’Sase (whoit, residues). The values have been calculated with the program IBSP (Kabscbh & Sander. 1983) using tetramrriv IVI’?(aw. .%rerages over a window of’ 5 residues are plotted.

(g) ( ‘rystul packing ~~60

aOr---

r50

Electron density (CT) Figure 7. Solvent statistics. The 369 solvent molecules are given as a hist,ogram using density intervals of 02% in the final (2F,-F,)exp(ia,) map. The average H-factor for all solvent molecules within each density interval is drawn as a continuous line.

The number of’ different contwt.s in NT’Xasr crystals is ra.ther small. For reference subunit 1. there exist four contacting neighbors in the cry&al. which were numbered IT through \‘. TWYI of thescb four contact,s are across dyads (I-11 arid I&ITT) and two conta,ots (T-TV and T-V) are mut’uallv identical (Table 6). Since ,VPXase forms tetratners ;n solution that are stable in the presence of I .3 M-urea (Yoole & Claiborne. 1989~~). these t,etramers have to IN, assigned in the crystal. As (*ontact i-11 is strong (Table 6) and relates to the dimer contact. ofC:Rase. there is no doubt that’ it is within a tetramer. Cont’aet I--III is the second one within the t,etramer. because the alternative assignment,. contact I--T\‘. leads to unlimited linear polymers of GRase-likp (horizontal chains dimer unit’s of dimers in Fig. g(a)). It is remarkable that the crystal packing contact I-TV is larger than the tetramer contact

Structure of NADH

Peroxidase

1335

Table 6 Crystal contacts Monomer packing interactionsa

Buried surface area (A2)b

Number of contacting atom pairs’

2900

773

I-1 I

I-111 I-IV. I-\”

460 620

75

109

Polar interactions Atom 1 Glul4-GE1 Asn21-GDl Lys51 -NZ Lysfl l-N2 Lys318-NZ Gln324-N Gly325-N Ser326-N Ser326-OG Rer326-OG Asn402-GDl Aer405-OG Phe424-0 Gln142NE2 HisWNE2 VallOl-N

Atom 2 : Trp432-NE1 : Lys444-NZ : Asp369-OD2 : Tyr419-OH : Glu415OEl : Asp421 -0D 1 :Asp421-ODl : Asp421-ODl : Asp421-0 : Asp421-ODI : Asn433-ND2 : Asp42 1-O : FAD-N3F : Va1145-0 : Thr213-OGl : Gln347-0

Donoracceptor distance (A) 306 305 2md 3.25

2+39* 297 3.06 302 338 29x 337 2.72

2.85 3.10 3.48 3.44

“The neighboring monomers 11 through V can be derived from the reference monomer I using the following rotations and translations in fractional co-ordinates:

%alculated with the program of Kabsch & Sander (1983). ‘Nonhydrogen atom pairs across the interface with distances less than 45 A. Contact I-II involves 72 residues (10. 14, 18, 21, 40, 42, 43, 45, 46, 51. 52, 57 to 60, 159, 163, 299 to 301, 303, 304. 307. 308. 311. 318. 323 to 330, 335,337,364,366 to 369, 396, 398 to 403,405 to 407,409,410,412,415, 417,419 to 428, 431 to 433. 440, 444, 447) and FAD. Contact I-111 involves 12 residues (47, 53, 55, 138. 141. 142. 145 to 147, 170 to 172). As these contacts are related by 2-fold axes. the residues listed here belong to both contacting monomers. Contact I-IV involves 25 residues (27, 72, 85 to 88, 99 to 101, 121. 180 to 1x2, 211, 213, 229, 230, 259, 260 9->->-? 274 275 315 347, 348, 350). where the underlined residues belong to monomer I and the others to monomer IV. %alt bridges. ‘These 2 contacts are identical.

I-III (Table 6). Given these contacts and the large channels that’ permit efficient substrate diffusion, it’ is conceivable t’hat the enzyme is crystalline in the bacterial cell. Unfortunately, we know of no observations concerning this hypothesis. A more distant look at, the crystal structure (in Figure 8(b) reveals that the tetramers are well fixed in a spacious (67% solvent) but obviously rigid (small R-factors) three-dimensional array. The packing arrangement gives rise to large solvent channels with diameters around 40 A. Such largechannels can affect crystal density determinations in gradients of organic solvents because these can diffuse quickly into the crystals. This problem may explain the results of Schiering et aE. (1989), who determined crystal densities corresponding to two instead of one monomer per asymmetric unit. (h) Chain fold comparison between NPXase

and GRase Between NPXase sequence homology

and GRase there of 21 o/o identical

exists a poor amino acids

(Ross & Claiborne, 1991), and the chain folds resemble each other indicating a distant evolutionary relationship. For determining the chain fold similarity in more detail, we superimposed the related dimers of NPXase and GRase (see section (g), above). An initial manual superposition of the 2-fold axes revealed substantial domain rearrangements. Therefore, a basic superposition was defined by running program OVERLAY (Kabsch, 1978) with a cutoff of 3 A for the c” atoms of the INTERFACE domain dimers of NPXase and GRase. As usual the program was started by select.ing an initial set of equivalent C” atoms visually. The final equivalenced set contained 2 x 81= 162 C” atoms, the superposition had a residual r.m.s. distance of 1.6 A. Subsequently, this superposition procedure was repeated with all domains separately. The equivalenced sets of c” atoms are given in Table 7; the chain fold superpositions are depicted in Figure 9. It turned out that all four domains superimpose with around 7O’j/b of their residues and that the residual r.m.s. C” distances range between 1.2 A and l.SA. Furthermore, the superpositions of each domain

1336

T. Stehle et al.

(a)

Figure 8. Crystal packing of the iWXase molecules. (a) Stereo view of the (1” backbone model of an WXase tetramrr with 2 additional subunits at bottom-left and top-right associated by contacts I-IV and I-V (contact surface I is in tetramer), respectively. Two dyads within neighboring tetramers are indicated. The dyads forming the large contact T--II and the small contact I-III lie vertically and horizontally, respectively. in the plane of the paper. (b) (la backbone drawing of 6 tetramers viewed along the c-axis, revealing large channels. The 2 tetramers in the middle sit above the others. The view is related to (a) by a 90” rotation around a horizontal axis in the plane of the paper. Among protein crystals, this packing is particularly space-demanding giving rise to V,= 3.8 ,h3/Da and a solvent content, of 67S,,.

revealed rotations of around 20” and translations around 4w of the first three domains along the chain relative to the basic superposition of the INTERFACE domain dimers. A superposition of single INTERFACE domains resulted in a rotation and translation of only 3” and 06 .& and an increase of the equivalenced set’ from 81 to 84 C” atoms (Table 7), demonstrating that not only the chain folds of the INTERFACE domains but also the

interfaces themselves are closely related in NPXase and GRase. Since the first three domains showed ximilal rotations, a further comparison was done with a combination of these domains. Here, the final equivalenced set was almost as large as the sum of the sets for single domains. and the residual r.m.s. (la distance was only slightly above the values for the separate comparisons (Table 7). Accordingly. t.hr

Structure of NADH

Peroxidase

1337

Table 7 Chain fold comparisons

FAD-binding NAD-binding CENTRAL INTERFACE

arrangements

of NPXase

and GRase

Fraction of equivalenced (I” atomsb (%)

Polar rotation angle’ (deg. )

72 105 57 84

64 78 75 67

18 21 19 3

2.7 5.5 4.7 0.6

1.4 1.4 1.2 I.6

227

70

18

40

1.5

Number of equivalenced C” atoms?

Domain

FAD-binding NAD-binding (IENTRAL

and domain

Translation distance (4

r.m.s. Cm deviation (4

plus plus

The superpositions were done as described in the text. “The C” atom reference sets used for the superposition with OVERLAY (Kabsch, 1978) contain residues: 2 to 20, 25 to 34, 36 to 38, 62 to 75, 77 to 81, 86 to 92, 99 to 112; 113 to 119, 121 to 125, 128 to 131. 149 to 189, 193 to 219, 224 to 229, 231 to 233, 235 to 246; 247 to 250, 255 to 283, 295 to 313, 317. 318. 320 to 322; 324 to 331, 333 to 342, 353 to 361, 372 to 393, 395 to 421. 423. 424, 433 to 438 of NPXase and residues 22 to 40, 43 to 55, 114 to 132, 137 to 157; 158 to 164, 169 to 177, 187 to 227, 230 to 256. 261 to 266, 277 to 279, 282 to 293; 294 to 297, 305 to 333, 335 to 353, 359, 360, 362 to 364: 366 to 383. 396 to 404, 416 to 464, 466, 467, 472 to 477 of GRase. bAs related to SPXase. ‘The polar rotation angle has been defined by Rossmann & Blow (1962). The rotation is relative to the superposition of the INTERFACE domain dimers.

domain rearrangement between NPXase and GRase consists of a concerted relative rotation of the first three domains by 18” and a translation of 4 A against the more or less identical dimer of TNTERFACE domains. It’ is tempting to speculate that this rearrangement is related to the extension of the FAD-binding domain around residue 60 of NPXase. As shown in Figure 9(a), this extension is much longer in GRase, where it forms a second interface across the molecular dyacl (Karplus & Schulz, 1987) pulling the first t,hree domains closer to this dyad. It should be noted that the act~ive center of GRase comprises the redox-active disulfide Cys58:Cys63 on the long extension as well as the INTERFACE domain of the other subunit with its catalytically essential residue His467’. Therefore, the second interface seems to be important in GRase because it keeps an exact geometric relation between the disulfide group and His467’ (Karplus & Schulz, 1989), thus tightening up the dimeric molecule. In contrast, no such requirement exists for NPXase where the active center is concentrated in one subunit, explaining the more open domain arrangement of this enzvme. For GRase, it had been pointed out that the FAD-binding and the NADP-binding domains are so similar that) a gene duplication is likely (Schulz, 1980). In order to establish this similarity for NPXase, we superimposed these two domains in the described manner and found an equivalenced set of 78 Ca atoms, which is 70% of the FAD-binding domain, and a residual r.m.s. C” distance of 1.5A. The superposition is depicted in Figure 10. For comparison, the respective superposition within GRase yielded an equivalenced set of 73 C” atoms with an r.m.s. C” distance of 1*2A. Since these

internal superpositions show differences in the same range as the superpositions of respective domains of NPXase and GRase (Table 7), we conclude that gene duplication, i.e. evolutionary relationship FAD-binding between and NAD(P)-binding domains is as likely as the relationship between NPXase and GRase.

(i) FAD as the first redox center The active center of NPXase can be subdivided into two redox centers, the isoalloxazine moiety of FAD and the Cys-SOH together with HislO. The sulfenic acid is placed at the &-side of isoalloxazine. As in GRase, the re-side of isoalloxazine is empty. Most probably, it accommodates the nicotinamide moiety of NADH as observed in the complex between NADPH and GRase (Karplus & Schulz, 1989). NPXase can bind NADH without, any steric hindrance in almost the same conformation as GRase binds NADPH. Moreover, NPXase has the usual NAD-binding site sequence fingerprint (Wierenga et al., 1986) at the correct place: Gly156Ser157-GZy158-Tyr159-Ilel6O-GZyl61. and Asp179 for hydrogen bonds to the adenine-ribose. In the unliganded structure of GRase (Karplus & Schulz, 1987), the re-side of isoalloxazine is shielded by a tyrosine side-chain (Tyr197). At the equivalent position NPXase has Tyr159. Assuming that the nicotinamide of NADH binds to NPXase as NADPH binds to GRase (Karplus & Schulz, 1989), Tyr159 has to swing out on NADH binding in order to avoid severe collision between its ring and the C-4 atom of the nicotinamide. As can be seen in Figure 11, the tyrosine hydroxyl group of NPXase is hydrogen bonded to the 0-4~~ atom of the isoalloxazine (Table 8), in contrast to GRase, where

1338

T. Stehle et al

lb)

ICI

Fig. 9.

the hydroxyl group of Tyr197 is close to the N- 10 atom. In NPXase, FAD mediates the reduction of the C’y&%sulfenic acid at the expense of two electrons taken from NADH. This corresponds with the role of FAD in GRase, where a disulfide group is reduced instead. This funckional simi1arit.y agrees well with

the st,ructural similarity betwern Sf’?(asr on on? hand and (:Rase and t,he family of rvlated disulfidr oxidoreductases on the other. A comparison of t hv FAl) conformat~ions in GKase and N PXase shows close resemblance. only one torsion angle (O-S’.-\PA-OAF-PF) differs by more than ‘LO-. As drmon&rated in the superposition of Figurv 1I’(a.). this

Structure of NADH

Peroxidase

1339

23

323

Figure 9. Ca backbone superpositions in stereo for separate domains of KPXase (thick lines) and GMase (thin lines). (a) FAD-binding domains together with FAD (Cys42 of NPXase is marked by a large dot. Cys58 and Cys63 of GRase are indicated by small dots), (b) NAD-binding domains together with FAD as a reference; (c) CETJTRAL domains, and (tl) INTERFACE domains, together with their dyads. With the exception of (a) where the molecule has been rotated by 30” around an axis vertical to the plane of the paper. the orientations are the same as in Fig. 3. The numbers denote the domain borders in WXase.

difference

relates

Phosphat,e

(PA)

to a 1 A shift of the adenylat’e in NPXase, whichis a caonsequence of steric hindrance caused by the sidechain of Ser9. This Ser9 is at a glycine position in the usual fingerprint sequence for FAD sites. The side-chain of Ser9 may be necessary in NPXase in order to stabilize the adjacent’ act,ive site base HislO. of’ FAU

Eggink

et al. (1990)

have

reported

an additional

sequence fingerprint for FAD-binding sites with a consensus sequence T-x-x-x-x-h-y-h-h-G-D, where h represents a small hydrophobic amino acid (Ile, Val, Ala or Leu) and y is an aromatic residue (Tyr, Phe or Trp). In NPXase this fingerprint is at positions 271 to 281 with the sequence Thr-Ser-Clu-Pro-AspVal-Phe-Ala-Val-Gly-Asp. In NPXase and in GRase

Figure 10. C” backbone superposition, in stereo, of the FAD- (thick lines) and the NAD-binding domains (thin lines) of NPXase. The orientation of the FAD-binding domain is the same as in Fig. 3. The superposition is based on the following equivalenced set of 78 C” atoms: 1 to 22, 25 to 39, 63 to 83, 87 to 92. 99 to 112 of the FAD-binding domain and 150 to 186, 198 to 218, 224 to 243 of the NAD-binding domain (see the text’).

1340

T. Stehle et al.

-

Figure 11. Superposition, in stereo, of NPXase and GRase at the NAD(P) binding sit,e. Depicted are the sequence fingerprint residues 154 to 163 of NPXase (thick lines) and residues 192 to 201 of GRase (thin lines) together with the isoalloxazines.

The superposition

is taken

from

the NAD-binding

domains

(see Table

7).

Table 8 Polar interactions FAD atom

NPXase atom

Donor-acceptor distance (A)

N-IA N-3A

79-N 33-N

2.98 3.14

N-7A N-6aA

Wat562 79-o Wat639 Wat562 Glu32-OE2

3.47 2.74 348 338 271

Glu32-OEI Wat687 Arg132-NH2 113-N Wat751 SerS-OG O-4’F Wat535 11-N Wat500 Serl lo-OG 281-N Wat532 OA2

265 3.44 282 341 2.73 2.73 2.75 3.20 312 2.69 2.73 280 2.84 2.75

Asp281-OD2 Cys42-OX2 299-N 299-N Wat452 424’.0 Tyr159-OH Wat490 Wat490

2.69 277 3.14 3.04 304 2x5 342 270 297

O-2’A 0-3’A O-Al

O-A2 O-F1

O-F2 0-4’F O-3’F 0-2’F N-IF O-2aF N-3F O-4aF N-5F

“Taken from Karplus

& Schulz (1987).

of FAD

in NPXase

Don.-fi.. .&cc. angle (deg.) 171 156 165 170 165 138 154 166 152 162 150 158 I -52 150 170 172 129 170 140

as compared GItase atom’

to GRase Donor-acceptor distance (A)

130-N 51-N 51.OG Wat326 130-O Asn129-ND1 Wat326 Glu50-OE2 Wat364 Glu50-OEI Wat54 Thr57-OG 1 WatlO

2.90 313 333 314 308 3.39 4-18 2.65 23 1 2.69 2.78 285 3.10

57-N O-4’F Wat359 3-N Wat4

3.16 2-96 2% 2.74 2.66

331-N Wat2 OA2 0-2’F wat359 Asp331-OD2 O-4’F 339-N 339-N Wat 15 467,-O I,ys66-N%

2.97 2.55 2.96 6.65 3.17 2.76 M.5 3.49 3.10 2.96 2.74 “,7X

Ly”66-XZ

301

Don:-H. .a density above the lo level. Conspicuously. there is no density at t.his level between the CA and CB atoms. This lack of density is possibly due to a Fourier series termination effect around the big sulfonic acid density because a test calculation damping this effect (by multiplying the structure factor amplit.udes in the resolution ranges 3.0 to 2%. 2% to 2.6. 2.6 to 2.4 and 2.4 to 2.16 wit’h factors 0%‘5. 0.70. O..% and 0.40, respectively) increased the density in thr CA-(I13 region. Conformational disorder due to the non-native sulfonic acid cannot be excluded. but seems unlikely because all atom mobilities of (-“ys(-SO,H)42 are similar and consistent with those of the neighboring residues: the H-fac+)rs of N. (‘A. (‘. 0, CB, SG. 0X1, OX2 and OX3 are 10. Il. 12. 12, 11. 1.5. 11. 13 and 13A*. respecti\-rly.

(k) Hydrogen

peroxide

access channel

The proposed H,O, access channel of NPXase is shown in Figure 14. The bottom of this channel is sealed by rather hydrophobic residues contributed by the second subunit. Close to the bottom there are Qs42 and the active-site base HislO. The inner surface of the channel is fairly polar and permits hydrogen bonding to substrate and products. Polar groups are SerS-OG, Phe39-0, Ser41-N, Ser41-OG. TyrSO-OH, OA2 from the adenylate phosphate and 0-2’F as well as 0-4’F from the ribityl moiety of FAD. The channel is partially filled with fixed water molecules, which probably point out the way for the entering substrate H,O, and the leaving produced water molecules. Tn conclusion NPXase shows us a new type of redox center that is structurally and functionally calosely related to the known redox centers of the disulfide oxidoreductases. Some of the struct,ural

Peroxidase

1343

differences like the domain find an explanation.

rearrangement

seem to

We thank Dr Keith Wilson and cao-workers at the EMBL outstation ab DESY, Hamburg. for their help in csollecting the synchrotron data. This work was supported by the Gradulertenkolleg Polymerwissenschaften (T.S. and G.E.S.) and by Xational Institutes of Health grant GM-35394 and a Grant-in-Aid from the Atnerican Heart Association (A.(‘., who is an Established Investigator of the American Heart Association). The atomic co-ordinates and the structure factors will be deposited in the Brookhaven Protein Data Bank under the accession number 1iWX.

References Ahmed. S. A. & Claiborne. A. (1989a). The streptococcal flavoprotein NADH oxidase. Evidence linking XADH oxidase and EADH peroxidase cysteinyl redox centers. J. Biol. (‘hem. 264. 198.X--19863. Ahmrd. S. A. & Claiborne. A. (1989b). The streptococcal flavoprotein h’ADH oxidase. Interaction of pyridine nucleotides with reduced and oxidized enzyme forms. J. Biol. Chem. 264, 19864-19870. Ahmed. S. A. 8r Claiborne, A. (1991). Artificial tiavins as probes of the active-site environment and redox behavior of the streptococcal 1;ADH peroxidase. In Flevins and Flawproteins lYY0 (Curt,i. B.. Ronrhi. S. & Zanetti. (i.. eds), pp. 659-662. de Gruyter. Berlin. Baker. E. R’. (1980). Structure of actinidin. after refinement at 1.7 A resolution. .I. Mol. Riol. 141. ‘M-484. Baker. E. N. &, Hubbard. R. E. (1984). Hydrogen bonding in globular proteins. Proyr. Hiophys. Mol. Biol. 44. 97-179. Hlevins, R. A. c1 Tulinsky. A. (1985). Comparison of the structures of dimeric solvent independent themselves and wit,h with z-chymotrypsin y-chymotrypsin. .J. Biol. (‘hem. 260. XX65-X8iZ. Kriinger. A. T., Kuriyan. J. & Karplus. Jl. (1987). C’rystallographic R-factor refinement by molecular dynamics. Science. 235, 458-460. (‘laiborne. A., Ahmed. S. A.. Ross. 1’. Jt Miller. H. (1991). The streptococcal NA DH peroxidasr and SADH oxidase: structural and mechanistic aspects. rn Flnvins and Flavoproteins 1990 ((‘nrti, B. Ronchi. S. 62 Zanetti. (i., eds). pp. 639-646. de Gruyter. Berlin. Davis, F. A.. Jenkins. L. A. & Billmers. R. L. (1986). Chemistry of sulfenic acids. Reason for the high by of sutfenic acids. Stabilization reactivit) and int,ramolrcular hydrogen bonding (‘hem. 51. elertronegat,ivity effects. J. Organic 1OX- 1040. dil)hosphopvridine Dolin. M. I. (1975). Reduced nucleotide peroxidase. J. Biol. C’hrm 250. 310-317. Dolin. M. I. (1982). XADH peroxidase of Streptococcus fawnlis. In Experiences in Biochumirnl Perception, (Ornston. I,. N. & Sligar. 8. (i.. rds). pp. P93&307, Acadetnic Press. iYew York. Eggink. G.. Engel, H., Friend. G., Terpstra. I’. & With&. B. (1990). Rubrrdoxin rrductasr of f-‘se~~domonas relationship to other oleoaoran,s: Structural flavoprotrin oxidoreductases based on one SAD and two F4D fingerprints. J. Mol. Biol. 212. 13.514Z. French. R. & Wilson. K. S. (1978). On the treatment of negative int,ensity observations. dcta (‘r,@allogr. sect. .4 I 34. .517-525.

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