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In this process FMN is first reduced in a reaction mechanism which is similar to that of other a-hydroxy acid oxidases (6, 7) including glycolate oxidase. However ...
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 5, Issue of February 15, pp. 3198-3207, 1991 Printed in U.S.A.

Spinach Glycolate Oxidase and Yeast Flavocytochrome bS Are Structurally Homologous and Evolutionarily Related Enzymeswith Distinctly Different Function andFlavin Mononucleotide Binding* (Received for publication, July 9, 1990)

Ylva Lindqvist and Carl-Ivar Branden From the Department ofMolecular Biology, Swedish University ofAgricultura1Sciences, BwMedical Centre, Box 590, S-752 24 Uppsala, Sweden

F. Scott Mathews From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 631 10

Florence Lederer From the Unite 25 de l’lnstitut National dela Sante et de la Recherche Medicale, Unite Associke 122 du Centre Natwnal dela Recherche Scientifique, Hhpital Necker, 75743 Paris Cedex 15, France

A comparison of the three-dimensional structuresof processmade up of twohalf-reactions. Inthefirsthalfthe flavin mononucleotide (FMN)-dependent enzymes reaction, the hydroxyl group of the substrate isoxidized and glycolateoxidase,flavocytochrome b2, and trimethFMN is reduced. In the second half-reaction, reduced FMN ylaminedehydrogenase is presented.Theirflavinis reoxidized by oxygen and hydrogen peroxide is produced. binding domains all have the same structural motif, The enzyme is widely distributed and has been studied the 8-fold O/a-barrel domain, whichis also present in mainly in mammals and plants. In green plants, glycolate a large number of other enzymes. FMN is bound in a oxidase is one of the key enzymes in photorespiration where similar fashion in all three enzymes. The binding site it oxidizes glycolate to glyoxylate (1).Net photosynthesis is is at the carboxyl-terminal endof the eight @-strands for carbon metabolism of the barrel where the active site is invariably found drastically reduced dueto this pathway which is initially catalyzed by the oxygenase reaction of in this typeof domain structure. The similarity of the structures of glycolate oxidase and flavocytochrome bz ribulose-1,5-bisphosphatecarboxylase/oxygenase (2). The x-ray structure of glycolate oxidase from spinach has extends to the loop regions and even outside the @/a: barrels with a root mean square deviation of 0.93 A been determined andrefined to 2 8, resolution (3). Theenzyme for 311 superimposed Ca-atoms and with a sequence molecule is octameric with 422 symmetry. The main part of identity of 37%. A detailed analysisof their active sites the subunit is folded into the common eight-stranded @/ashows, however,that the orientationof FMN is signif- barrel motif. This domain binds thecofactor FMN and contains a 29-residue loop covering the active sitecleft where the icantly different in the two structures due to different conformations of residues in the end of strand one. substrate is bound. In addition, the first 70 residues of this small helical domain Thus, in flavocytochrome bz a hydrogen bondis formed 369 residuelongpolypeptideforma between the FMN N-5 position and the main chain which partially covers the C-terminal endof the @/a-barrel. amide of Ala-198, while in glycolate oxidase, the ring Flavocytochrome bz from yeast (EC 1.1.2.3) is a tetrameric system is tilted awayfrom the strand, creating a pocket enzyme, which carriesoneFMNandoneprotohemeIX/ on the re-sideof the FMN ring wherea water molecule subunit of M , 57,000 (4,5). It islocated in the intermembrane is bound. Model building shows that this site could space of mitochondria and catalyzes the oxidation of lactate accommodate the hydroperoxidemoiety of a FMN-4a- to pyruvate. In this process FMN is first reduced in areaction hydroperoxide intermediate. Thus, in the course of mechanism which is similar to that of other a-hydroxy acid evolution, a few mutations in, andclose to, the active oxidases (6, 7) including glycolate oxidase. However, in flavsites have fine tuned these enzymes to exert their spe- ocytochrome bl, FMNH, is reoxidized by transferringits cific functions as a n oxidase or transferase, respec- electrons through the heme group t o cytochrome c which is tively. thenaturalelectronacceptor (5). This sequence of steps involves the transient formationof a catalytically competent flavin semiquinone(8). The x-ray structure of flavocytphrome bz frombaker’s Glycolate oxidase (EC 1.1.3.15) is a peroxisomal enzyme of M , 40,00O/subunit that catalyzes the oxidation of a-hydroxy yeast has been determined to 2.4-A resolution and has been residues form aseparate hemeacids by using flavin mononucleotide, FMN,’ as cofactor in a refined (9). The N-terminal99 binding domain, thecytochrome bl core, which is homologous b6 (10,ll).The follow* This work was supported by Swedish Natural Science Research and structurally similar to cytochrome Council Grant 4030-3, by United States Public Health Service Grant ing 365 residues were shown to fold into an eight-stranded@/ 20530, and by a North Atlantic Treaty Organization travel grant. a-barrel that binds FMN, preceded by a small helical domain, The costs of publication of this article were defrayed in part by the thus similar to the glycolate oxidase structure. Finally, a 25payment of page charges. This articlemust therefore be hereby residue extended tail makes contact with each of the other marked “advertisement” in accordance with 18 U.S.C. Section 1734 three subunitsof the tetramer. solely to indicate this fact. The sequence of glycolate oxidase (12, 13) exhibits 37% The abbreviations used are: FMN, flavin mononucleotide; r.m.s., identitytothe sequence of the flavin-binding domain in root mean square.

3198

Comparison of Glycolate Oxidase and Flavocytochrome b2

3199

FIG.1. Diagram illustrating similarities and differencesin F M N binding to glycolate oxidase (Mue) and trimethylamine dehydrogenase (red).The Ca-atoms of the @-strands after superposition of the O/a-barrels are shown.

flavocytochrome b2 (14, 15), thus indicating that the two proteins are homologous. In this paper we present a detailed structural comparison of these two proteins and show that although they show a very high extent of structural similarity (0.93A r.m.s. deviation for 311 Ca-atoms), there is a distinct difference in their FMN binding which might constitute the basis of their different reoxidation modes. We have also made a comparison with a thud FMN-containing enzyme, trimethylamine dehydrogenase (EC1.5.99.7)whose structure has been determined (16).The subunit M,is 86,000, and it is composed of three structural domains of which the largest, N-terminal domain, is a Pla-barrel that binds FMN and an iron-sulfur cluster [4Fe-4S]. No chemical sequence is available for this protein. MATERIALS AND METHODS

A detailed description of the active site (17)and of the complete refined structure (3) of glycolate oxidase has been published. The model coordinates used in this study have a crystallographic R-value of 18.9% to 2 A. The refined structure of flavocytochromebz has also been described (9).The R-value is 18.5% to 2.4 A for this model. This structure has two crystallographically non-equivalent subunits related by a local 4fold axis which is coincident with a crystallographic 2-fold axis. One of these has an ordered cytochrome domain while in the other the cytochrome domain is disordered. In addition, the latter domain contains a molecule of pyruvate bound in the active site while the former does not. We have used that subunit which has the ordered cytochrome domain for most of the comparisons, but all results are equally valid for comparisons made with the other subunit. The coordinates used for trimethylamine dehydrogenasewere those obtained from preliminary refinement at 2.4 A resolution ( R = 22.4%)' based on an x-ray sequence (18). The comparisons of Ca-atoms between glycolate oxidase and flavFIG.2. SuperpositionoftheCa-atoms of onesubunitof flavocytochromebn (yellow)on glycolate oxidase (Mue).

F.S. Mathews, unpublished observations.

Comparison of Glycolate Oxidase and Flavocytochromeb2

3200 PCB FCB

Gox

51

1 E P K L D M N K Q K 61 I F E P L H A P N V .

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121

11 21 31 41 51 I S P A E V A K H N K P D D C W V V I N G Y V Y D L T R F L P N E P G G Q D V I K F N A G K D V T A 71 81 91 101 111 I D K Y I A P E K K L G P L Q G S M P P E L V C P P Y A P G E T K E D I A R K E Q L K S L L P P L D M 131 171

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315 335 K A G P K A M K K T N V E " " " E S Q G A S R A L S K F I D P S L T W K D I E E L K K P F L T L K N F E G I D L G K M D K A N D S G L S S Y V A G Q I D R S L S H K D V A W L Q T 176 216 1 1 " " a """I I a4 355 395 E D V I K A A E I G V S G V V L S L D F S R A P I E V L A E T M P I L E Q R N E D A R L A V Q H G A A G I I V S L~ D ~Y ~V P A T I M A L E E V V K A A Q G - - 236 276 256 266 a5 _ _ _ _ _ _ _ I (--_-_____ a6 I- 86 --I I 415 425 455 4 65 435 445 C T D V ~ K A L C L G A K G V G L G R P F L Y A N S C Y G R N G V E K A I E I L R D E I E G T D V F K A L A L G A A G V F I G R P V V F S L A A E G E A G V K X V L Q M M 291 301 311 321 331 1""a7 I 1"I I""--@ """"I I"""""""""_ a8 475 P D L L D L S T L K A R T V G V P N D V L Y N E V Y E G P T L T E F E D A R S H I A A D W D G P S S R A V A R L - - - - - - - - - - - - - - - - - 351 361 I- pD I

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K T K L P I V I X V Q R T I T S L P I L V K V I T A 345 226 I I- p5 --I I405 L K D K L E V ~ V D G G V R R - - R I P V ~ L D G G V R R 281 I- p7 "I

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3'10. 3. Sequence alignment of flavocytochrome & and glycolate oxidase based on the structural alignment. Identical residues are indicated in boldfaced type.The secondary structure elements are indicated, and residues in the active site involved in substrate or cofactor binding or catalysis are boxed. The placement of a deletion between flavocytochrome b2 positions 313 and 314 is arbitrary since there is no sequence or structural similarity between positions 300-324.

ocytochrome b2 were made, and the superimposed structures inspected, using the graphics program 0 (19). Atoms were considered to superimpose if they are in a stretch of at least 3 residues with a distance between equivalent atoms of less than 2.6 A. The comparison to trimethylamine dehydrogenase was made on Ca-atoms using the program HOMO (20). The initial superposition was made from 4 residues for each &strand in the barrel, i.e. 32 Caatoms. RESULTS AND DISCUSSION

@/a-Barrel Similarity between Trimethylamine Dehydrogenase and Glycolate Oxidase-The eight-stranded p/Blcw-barrel structure is a common structural motif that has been found in a variety of different enzymes. This motif occurs among enzymes of non-homologous amino acid sequences and quite different functions. They are built up from a common coreof eight parallel strands and eight helices connected by loop regions that vary considerably in length and conformation. When these structures of non-homologous sequences are superimposed and compared it is found (21) that the common core comprises about 160 residues with an r.m.8. deviation of around 3.0 A in their Ca-atompositions. When the flavin-binding domain of trimethylamine dehydrogenase was compared with glycolate oxidase, maximizing the number of overlaps and minimizing the r.m.8. deviation, FIG. 4. Dingram illustrating similarities and differences in Z+, (yeuow).The Ca-atoms of the &strands after superposition of shown. FMN binding to glycolate oxidase (Mue) and flavocytochrome the @labarrels are

and Flavocytochrome bz

Comparison of Glycolate Oxidase

3201

the number of eguivalenced Ca-atoms was 168 with an r.m.s. lie also in the @/&-barrel core, but in addition, both structures deviation of 2.6 A. This is slightly closerthan what is obtained have two short anti-parallel strands closing off the barrel at comparing glycolate oxidase and triosephospha!e isomerase, the N-terminal end of the strands of the barrel. The FMN 167 equivalences with an r.m.s deviation of 2.9 A (22). In the molecules are positioned in a similar wfy in both enzymes latter case the similaritybetween the structuresresides in the (Fig. 1) with an r.m.s. deviation of 1.6 A when the rotation used. The core of the @/a-structure,i.e. in the eight strands andhelices, matrix obtained by superimposing the Ca-atoms is while the connectingloops are quite different. The similaritiesbinding site is at the carboxyl-terminal of the 8-strands of between trimethylamine dehydrogenase and glycolate oxidase the barrel in the concave surface formed where the strands are turning outward toward thehelices. The N-5 edge of the FMN-ring is directed outward, whereas the ribityl side chain is buried inside the barrel in both structures. In trimethylamine dehydrogenase the FMN molecule is covalently bound through its 6 position to a cysteine side chain of the protein. The phosphategroups are in bothcases in approximately the same position as the substrate phosphate group in triosephosphate isomerase, at the N-terminal endof an a-helix in one of the loops that connects the strandswith the helices. Thus thefunctionalbasis for FMNbinding involves thesame FIG.5. Schematic diagram illustrating the relative posi- folding framework but does not require extensive structural similarity between thebarrelstructures.No chemicalsetions of residues in the interior of the B/a-barrel in glycolate oxidase and flavocytochrome ba. This hydrophobic core is formed quence is known for trimethylamive dehydrogenase, but with the r.m.s. deviation obtained (2.6 A) we do not expect to find from side chains in the 8-strands pointing toward the center of the barrel. significant sequence homology between these two enzymes.

Strand 2

Stran

FIG.6. Stereo diagram of the side chain packing of residues in the 8strands pointing in toward the center of the barrel. The view is down the barrel axis from the carboxyl end of the strands in glycolate oxidase (a)and Bavocytochrome bp( b ) .

Strand 7

Strand 1

b

Strand 1

:Kana 1

Strand 7

TABLE I Corresponding residues in the hydrophobic layer between the fi-strands and a-helices of the fila-barrel in glycolate oxidaseand flavocytochromeb2 82

Pl

Glycolate oxidase Flavocytochrome b,

I1 FV QYL 01

Glycolate oxidase Flavocytochrome b,

ETAA LVA EVAC

ML I L QI a2

VVA PI1 WILK

84

I33

R F L VI a3

LV a8

a4

TLVVA AA WVLQ LVV

VL

05

86

IV I1

IV VL

a7 a5

VA

87

88

vv

VL

VA VA

VMF ALI

a6

PLTML

Comparison of Glycolate Oxidase and Flavocytochromebz

3202

TABLEI1 4-fold contacts in glycolate oxidase and flavocytochrome b:! Structurally equivalent interactions are aligned in the two columns. SS, side chain to side chain interaction; MS, main chain toside chain interaction; MM, main chain to main chain interaction. Glycolate oxidase

Flavocytochrome 6%

E-20 - K-169 NZ

MS

T-40g - E-165 Oe T-40g - K-169 NZ T-40 - R-163 NH N-5Nd - E-165 Oe E-80e - K-169 Nz S-430g - Y-261 OH F-47N - Y-261 0 R-48NH - E-271 Oe R-48NH - E-236 Oe

ss ss MS ss ss ss MM ss ss ss

R-50NH - D-237 Od I-51N - D-237 Od

MS

I-53N - L-211 0 V-55N - R-209 0 R-290NH - D-31 Od R-290NH - E-165 Oe R-290NH - E-30 Oe

MM MM

ss ss ss ss ss

E-3330e - R-163 NH E-3350e - R-160 NH

Tyr 143

a

Lys 349

His373

Tyr 24

b

Lyr 230

His 254

FIG.7. Schematic diagram of the active site residues and substrates. The flavin is in a plane below the ligand and residues His-254/373, Asp-157/282, Arg-257/376, Tyr-24/143, and Tyr-129/ 254. a, pyruvate bound to flavocytochrome bp; b, glycolate modeled into the active site of glycolate oxidase.

N-121 Nd - F-297 0

SM

1-123 0 - Q-288 Ne N-124 Nd - E-290 Oe D-127 Od - K-294 NZ

MS

F-166 N - F-380 0 K-167 Nz - E-390 Oe

MM

P-161 0 - R-353 NH

MS

1-170 N - D-356 Od L-171 0 - W-332 Ne V-174 N - P-328 0

MS MS MM

R-414 NH - D-150 Od R-414 NH - E-290 Oe

ss ss ss

D-456 Od - R-320 NH N-492 Od - H-162 Nd N-492 0 - R-155 NH D-493 Od - H-159 Nd D-493 Od - H-162 Ne D-493 Od - Y-126 OH Y-500 OH - R-155 NH Y-500 OH - E-151 Oe E-501 Oe - W-141 Ne T-506 N - D-127 Od E-509 Oe - L-115 N

ss ss ss

ss MS ss ss ss ss ss ss MS SM

The gross features of the position and orientation of the bound coenzyme FMN with respect to the barrel structures are thus similar for the trimethylamine dehydrogenase and glycolate oxidase flavin domains. When the main chain frameworks are superimposed, the FMN molecules are also approximately superimposed. This situation is reminiscent of the binding of another coenzyme, NAD,to theclassical nucleotide binding motif (23, 24). In that case if was found that NAD bound in a very similar orientation to the coenzyme-binding domains of alcohol-, lactate-, and glyceraldehyde phosphate dehydrogenase despite their widely different amino acid sequences. The flavin-binding domains compared here thus provide a second example that, when similar polypeptide frameworks are used to bind the same large flexible molecule such as acoenzyme, this molecule binds in a very similar way in spite of completely different amino acid sequences of the domains. Structural Comparison of Glycolate Oxidase and Flauocytochrome b2-When we superimposed flavocytochrome bz with glycolate oxidase in a similar way we obtained 311 equivalent Ca-atoms with an r.m.s. deviation of 0.93 A. This implies that the three-dimensional structures are largely identical. Thus, not only the common core, but also virtually all loop regions exhibit very similar conformations. Even the N-terminal regions (residues 1-70 in glycolate oxidase, 123-192 in flavocytochrome b,) which form part of the small additional helical domain outside the barrel superimpose well. Inspection of the superimposed structures (Fig. 2) shows that the only large significant difference is that the loop in glycolate oxidase between strand four and helix four of the barrel, involving 29 residues which cover the active site, is moved away from the active site in flavocytochrome b, and is replaced by the cyto-

Comparison of Glycolate Oxidase and Flavocytochromebz

3203

FIG. 8. a, stereo diagram of the SUperimposed active sites of glycolate oxidase (blue) and flavocytochrome b2 (yellow). The small sphere behind the flavin is a buried water molecule in glycolate oxidase which is not present in flavocytochrome b,. b, schematic drawing of a with labels.

b

Trp 108/Leu 230

Gln 127/252 Tyr 1291254% Thr 155/280

& -.J

Lys 2301349

Asp 1571282

chrome domain. Other, minor differences between the two structures are that this loop is 5 residues shorter in flavocytochrome b2,that helix six of the barrel has a slightly different tilt, and that three loops at the N-terminal end of the 0strands p2, p3, and 07 in the barrel are 2-5 residues longer in flavocytochrome b2. Theseinserted residues in flavocytochrome bz are not involved in any packing interactions, and the affected loops are in both structures facing the solvent and are of no functional significance. Flavocytochrome b2 has also a 1-residue insertion at the beginning of helix 1and its extended tail of about 25 residues at theC-terminal end does not exist in glycolate oxidase. A sequence alignment based on

Tyr 24/143

frg

251/316

His 2541373

the structural alignment is shown in Fig. 3. A striking observation is that in spite of this strong similarity in the backbone structure, the FMN orientation and binding mode in the two structures, although similar, are significantly different. The r.m.s. deviatipn between the atoms of the bound FMN molecules is 1.1 A using the rotation matrix obtained by superimposing the Ca-atoms (Fig. 4) while the r.m.s. deviatiop for the 32 Ca-atoms in the S-strands of the barrel is 0.57 A and for 12 residues in the loops involved in FMN binding or catalysis 0.73 A. If the main chain and Cp-atoms of 10 residues, (Tyr-24/143, Ala-76/Ser-195, Ser106/228, Gln-127/252, Tyr-1291254, Thr-155/280, Asp-1571

3204

Comparison of Glycolate Oxidase and Flavocytochrome bz

282, Lys-230/349, His-254/373, and Arg-257/376) is used for superposition of the active sites, theirr.m.s. deviation is 0.57 A. The r.m.s for FMN in this superposition still is 0.90 A with 1.1 A obtained for the atoms in the ring system while the ribose and phosphate atoms gave an r.m.s. distance of0.54 i.e. it is mainly the flavin ring which is in different orientation in thetwo structures. It might be expected that thevery high structural similarity of the two P/a-barrels might require a higher than average degree of sequence conservation among the side chains packed in the interior. However, excluding residues at the carboxylterminal end of the strands that are involved in enzymatic function, thesequence homology of the strandresidues in the hydrophobic interior of the barrel is not higher than in the rest of the sequence (Fig. 5). The sequence differences in this hydrophobic core have been accommodated without perturbing the backbone structure. A thorough inspection of the side chain packing shows (Fig. 6) that this isgenerally not due to simple compensating substitutions but to amore complex series of replacements. For example, the simplest setof compensatingsubstitutionsarefoundin a region of the core occupied by the side chains of Phe-306, Met-74, and Thr-104 in glycolate oxidase. Corresponding residues in flavocytochrome b2 are Gly-430, Tyr-193, and Met-226, respectively. In thehydrophobic layer between the p-strands and theahelices, the sequence conservation of residuesfrom the 0strands is actually lower than the average between the two structures, with 24% identity compared with 37% overall. It

is, however, slightly higher than average, 42% for residues from the helices (Table I). This leads to an overall sequence identity of 35% in this second hydrophobic core. The packing of the subunits of the two enzymes has also been compared. The 4-fold packing arrangement, employing a crystallographic axis in the case of glycolate oxidase and a local axis in flavocytochrome b2, is very similar in the two enzymes. Superposition of half the tetramer of flavocyto5hrome b2 on glycolate oxidase gave an r.m.s. deviation of 0.98 A for 616 atoms. The interactionsmade in this packingwere analyzed and compared (Table11).This comparison revealed that many interactions areconserved, but there arealso quite a few that are unique to the respective enzymes. In particular there are very tight subunit contacts made around the 4-fold axis in flavocytochrome b2 by the tail region which does not exist in glycolate oxidase. Ontheotherhand,theother subunit interactions inglycolate oxidase about the%fold axes normal to the 4-fold axis, which formthe octameric structure, are not conserved in flavocytochrome bp. Active Site Geometry andSubstrate Binding-The substrates for glycolate oxidase and flavocytochrome bz are very similar, both being small a-hydroxy acids, glycolate and lactate, respectively. In flavocytochrome b2 we have observed a bound molecule of pyruvate, the productof lactate oxidation, to one of the subunits (9) (Fig. 7a). In this subunit theflavin is in the semiquinone form. The carboxyl group of pyruvate binds toArg-376 and Tyr-143 and the keto group is hydrogen bonded to Tyr-254 and His-373. The anionic form of the

FIG. 9. Stereodiagram of FMN and loop 1 in glycolate oxidase (a) and flavocytochromeb z ( b ) .Note the difference in main chain conformation resultingin ahydrogenbondbetween NH-198andFMN-N-5in flavocytochrome b, which is absent in glycolate oxidase.

$Yu-436 .-4 3 6

Comparison of Glycolate Oxidase flavin semiquinone, with thenegative charge of deprotonated atom N1 distributed over the Nl-CZ = 0 locus, is stabilized by ionic interactions to a buried Lys-349 near the N-1, 0-2, position. In glycolate oxidase we have built a model for binding of the substrate to the oxidized enzyme (Fig. 76) by assuming that the position of two bound water molecules in the active site might simulate the positionsof the two oxygen atoms of the carboxyl group of glycolate (17). In fact,using the superposition of the Ca-atoms, the glycolate model falls on top of the experimentally observed pyruvate in flavocytochrome b,. The bindingof the carboxylateagain occursthrough Arg-257/ 376 and Tyr-24/143. The substrate hydroxyl is now oriented toward the hydroxyl group of Tyr-129/254 as proposed for lactate in theflavocytochrome b, active sites (25, 26, 30). The backbone atoms of the active site residues in the two structures superimpose strictlyand alsomost of the side chains have virtually the same conformation (Fig. 8). There is only one sidechain in the active site which is not conserved, Trp-108 in glycolate oxidase which is Leu-230 in flavocytochrome bO. Part of the volume of thetryptophan ring is occupied by the propionate side chain of the heme in flavocytochrome b,. In loop 1 close to the active site, Pro-77 in glycolate oxidase is Ala-196 in flavocytochrome b,, but the main chain conformations of these residues are the same. One important difference in FMN binding is a hydrogen bond from the side chain of Ser-195 in p-strand 1 to the ribityl side chain in flavocytochrome b2 (Figs. 9and lo), which is not present in glycolate oxidase where the corresponding residue is Ala-76. This difference in hydrogen bonding contributes to the ease withwhich FMN can be abstracted from glycolate oxidase by raising the salt concentration as compared to flavocytochrome b,. A second difference in loop 1 is the main chain conformations of Thr-78/197 which has a d,$-value in the high energy region of the Ramachandran plot in the case of glycolate oxidase. The side chain conformationsof these threonines are also different in the two structures. This might be caused by Leu-436 in flavocytochrome bz, which corresponds toVal-312 in glycolate oxidase. This residue could restrain the position of the methyl group of Thr-197 in flavocytochrome bZ (Fig. 9). The only other difference in the vicinity of this Thr is Gln-81 in glycolate oxidase which corresponds to Cys-200 in flavocytochrome 21,.The difference in conformation of Thr78/197 gives rise to a hydrogen bond between NH-198 and N-5 of FMN in flavocytochrome bp and is probably the main factor influencing the different orientationsof the flavin ring in the two structures. The conformationof all other residues in glycolate oxidase that are strained, with d,$-values in the high energy region of the Ramachandran plot, are conserved in flavocytochrome b,. All of these residues are important for the geometry of the active site,i.e. for function (3). The third majordifferencein the active site is a water molecule, close to the 0-4,C-4 positions of the isoalloxazine ring (Figs. 8 and 9) inglycolate oxidase, which is not present in flavocytochrome b,. In the latter, where the FMN ring system lies closer to strand one and the amide of Ala-198 forms a hydrogen bond to theflavin N-5 there is no space for a water molecule. Table I11 shows the distancesfor the various polar interactionsfor FMN in thetwo proteins. The different locations of the flavin rings are also reflected in a slight but pronounced perturbationin the same directionof some of the side chains (mainly Tyr-128/254, His-254/373, and Lys-230/ 349) around the FMN, resulting in a slightly different hydro10). gen bond network in the active site (Fig. Mechanistic Implications-The chemical mechanism of the oxidation of a-hydroxy and a-amino acid substrates by fla-

Flavocytochrome and

b2

3205

voproteinshasbeenstudiedindetailinrecentyears (for reviews, see 6, 26-29). It is generally agreed that the first step in the substrate oxidative half-reaction is abstraction of a proton from the carbon atom adjacent to the carboxylate, the a-carbon, with formation of a carbanion. The probable glycolate/lactate-binding mode described above is compatible with thisso-called carbanion mechanism, in that itplaces the a-hydrogen in a suitable orientation for attack by atom N-3 of His-254/373 (17, 25, 26, 30). Studies with the flavocytochrome b, mutant Tyr-254-Phe also support the structure proposed for the Michaelis complex (30). As discussed before (6,27,28), the next step in reductive the half-reaction, namely electrontransfer from the carbanion to flavin N-5, could occur either through transient formation of a covalent bond between the substrate and FMN N-5 or through thesuccessive transfer of two single electrons. Although specific covalent intermediates were observed with D-amino acid oxidase (31) and Mycobacterium lactate oxidase (32),the generality of covalent adduct formation in normal catalysis remains to be established. No positive evidence concerning this point was obtained with glycolate oxidase (33) or flavocytochrome bz (34). Modeling studies at the active site of flavocytochrome

mi254

b

Ser 228

Hemepropionate I

His.373

FIG. 10. Hydrogen bond network in the active site of glycolate oxidase (a)and flavocytochrome ba ( b ) (subunit with heme-binding domain).

Comparison of Glycolate Oxidase Flavocytochrome and

3206

bz

TABLE I11 Polar contacts with FMN in glycolate oxidase and flavocytochrome b, FMN oxidase

Glycolate

Flavocytochrome bl

Protein

Subunit 1

2.8

3.5

3.7

Nz N1 02 02 N3 N3 N3 04 04 04 04 N5 N5 02* 02* 02* 03* 03* 03* 03* OPI OP1 OP2 OP2 OP2 OP3 OP3 OP3

K230/349 Nz K230/349 T155/280 Og Q127/252 Oe

2.8 3.0

Water

T155/280 Og Sl06/228 Og

3.0 2.9 2.7 4.0 3.3 3.2 4.6

Water Water Amide 79/198 N 3.0 Water 2.3

3.5 3.2 2.7

Water R289/313 N H R289/313 N H Amide 287/411 N 3.2

4.5 2.7

Subunit 2

2.8 2.8

4.5 2.6

2.8

Y129/254 OH

Nz K230/349 Carbonyl 77/196 0 2.9 S 195 Og D 285/409 Odl D 285/409 Od2 K 230/349 Nz S 252/371 Og 3.5 Amide 309/433 N R 309/433 N H Amide 308/432 N R289/413 N H

2.9 2.6 2.8 3.1

2.7

2.8 2.7 5.2 3.0 3.3 2.7 2.7 3.3 3.1 3.2 2.8 3.3 2.8 3.2 2.5 3.1 2.8 2.8

4.2

4.1

3.2

3.0

3.2 2.9 2.7 3.0 3.7

2.8 2.8 3.1

3.1 3.0 3.0 3.0 2.7 3.3 2.6

2.8 3.2 2.6 2.9

b, have suggested the possible occurrence of steric hindrance 0 HlC I 'CHI in the putative covalent intermediate, in particular due to interference by the methyl group of Ala-198 (26). In this respect, crowding of a lactate covalent intermediate due to 0 the methyl group of homologous Ala-79 would be less severe R a 3 a b inthe glycolate oxidase activesite. Due tothedifferent orientations of FMN, N-5 is further away from this residue FIG. 11. a, flavin la-hydroperoxide.O,, proximal oxygen;Od, distal a than it is in flavocytochrome b,. With the smaller substrate oxygen. b, 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin, glycolate, nothing seems to prevent the formation of a cova- model for flavin 4a- hydroperoxide. lent intermediate. Thus, the similarities at the active sites support the idea of an identical first step, carbanion formation, for the chemical reaction catalyzed by the two enzymes. change around the FMN was observed at 3-A resolution upon They do not give any evidence as to the nature of the second flavocytochrome b, oxidation(38),thusspeakingstrongly step, electron transfer proper, and whether it is identical or against this hypothesis, it cannot yet beprecluded that such not in thetwo classes of enzymes (electron transferaseversus a rearrangement takes place in the caseof glycolate oxidase. oxidase). The present structure comparison suggests an alternative On the other hand the flavin oxidative half-reaction is very hypothesis, namely that the water pocketclose to the 0-4,Cdifferent in the two enzymes, and we believe the differences 4 positions of the isoalloxazine ring on the re-side (Figs. 8 incofactor binding in the N-3,C-4,0-4,N-5 area are quite and 10) in the oxidized enzyme is the oxygen pocket of the revealing in this respect. It was earlier suggested (17) that reduced enzyme. The water molecule, which makes several flavin reduction was accompanied by a bending of the flavin hydrogen bonds, couldbe replaced by oxygen, which would be ring and that the water molecule close to the 0-4,C-4 position suitably locatedforreceiving electrons from FMNHz and of the FMN ring in glycolate oxidase could be replaced by the forming a covalent bond at position C-4a. It is premature to 0 - 4 atom of FMN in the reduced enzyme. In this position, speculate as to which groups might orientdioxygen by hydrothe 0-4 atom would make new hydrogen bonds, compensating gen bonding to it and might possibly protonate the distal for those lost on bendingof the ring. Attack of oxygen on C- oxygen of the hydroperoxide, or as to what would cause the 4a and formation of a hypothetical transient C4a-hydroper- collapse of the compound to oxidized flavin and hydrogen oxide (35-37) would then orient the latter on the si-face of peroxide. At any rate, thepyrimidine ring of the cofactor, in the flavin toward His-254. The difference in FMN orientation glycolate oxidase, is not tooclose to the backbone to prevent between the twoenzymescould thus be explained by the oxygen binding; the re-side in the N-3,C-4,0-4 area appears different redox states of the cofactors in the crystal structures more open to solvent and hence to oxygen than would be the of glycolate oxidase (oxidized) and flavocytochrome b, (re- si-side with the product bound to it. This observationmay be duced). The difference in FMN orientation in the two struc- important since it has beenshown with several oxidases that tures cannot however be accounted for solely by changing the reaction of the reduced enzyme-product complex with oxygen bent FMN in flavocytochrome b, into a flat conformation, is faster than that of reduced enzyme alone (39, 40). Unforbut the whole ring system has to move considerably relative tunately, the case has not been studied with glycolate oxidase. to the ribose side chain. Although no such conformational In contrast, in flavocytochrome bp,solvent and hence oxygen

Comparison of Glycolate Oxidase and FlavocytochromebZ

3207

FIG. 12. Stereoview of FMN in glycolate oxidase (thin line) superimposed on 4a,5-epoxyethano-3-

methyl-4a,5-dihydro lumiflavin (thick line).

L

a access to the flavinre-side is prohibited by the hydrogen bonds from the side chains of Gln-252 and Ser-228 to FMN N-3 and 0-4,respectively, and from the main chain nitrogen of Ala-198 to N-5 (Fig. 106). If this hypothesisis correct, thecofactor in glycolate oxidase would receive electrons from the substrate on the si-side and deliver them to oxygen on the re-side. There cannot be any objection a priori to this transfersince no oxygen is incorporated into the productby glycolate oxidase (41).Interestingly enough,inp-hydroxybenzoate hydroxylase, oxygen isattacked also from the flavin re-side, but this isalso the side to which the reductant NADPH and the hydroxylatable substrate bind (42, 43). A support of this hypothesis is that the structureof 4a,5epoxy ethano-3-methyl-4a,5-dihydrolumiflavin(44) (Fig. l l ) , an analogue of the flavin-4a-hydroperoxide intermediate, is the enantiomer that orients what corresponds to the hydroperoxide part on the re-face. Superposition of this analogue on FMN in glycolate oxidase (Fig. 12) shows that the hydroperoxide would fit excellently in the water pocket with the distal oxygen in hydrogen bond distance to main chain atom 0-78, FMN 0-2, and 0-7 of Ser-106. With FMN in this conformation an even better access to the suggested oxygen pocket from the outside is obtained. A corresponding structure for the 4a-hydroperoxide intermediate in p-hydroxybenzoate hydroxylase has been suggested (43). The difference in FMN orientation in the two structures would thus be the causeof the functional difference between the enzymes in the flavin oxidative half-reaction. The emergence of the flavooxidase and flavodehydrogenase function have proceeded by divergent evolution from a common precursor. During this evolutionarily process, the occurrenceof a few mutations has had a decisive influence on the conformation of Thr-78/197 and thus the orientation of the FMN ring system, determining the ultimate functionof these enzymes. The gene fusion which linked the cytochrome b2 domain to the flavodehydrogenase ensured efficient delivery of electrons to thephysiological acceptor, since it appears that isolated the flavodehydrogenase domain does not react with the isolated hemoprotein (45).3 REFERENCES 1. Frigerio, N. A., and Harhury, H. A. (1985) J. Biol. Chem. 2 3 1 , 135-155 2. Miziorko, H. M., and Lorlmer, G. (1983) Annu. Rev. Biochem. 52,507-535

S. K. Chapman and G. A. Reid, personal communication.

3. 4. 5. 6. 7. 8. 9. 10. 11.

b Lindqvist, Y. (1989) J. Mol. Biol. 2 0 9 , 151-166 Jacq, C., and Lederer, F. (1974) Eur. J. Biochem. 4 1 , 311-320 Appelhy, C. A,, and Morton, R. K. (1954) Nature 1 7 3 , 749-752 Ghisla, S. (1982) in Flavins and Flavoproteins (Massey, V., and Williams, C. H., eds) pp. 133-142, Elsevier North Holland, Inc., Amsterdam Urban, P., and Lederer, F. (1985) J. Biol. Chem. 2 6 0 , 11115-11122 Capeillere-Blandin, C., Bray, R. C. Iwatsuho, M., and Labeyrie, F. (1975) Eur. J. Biochem. 54,549-566 Xia, Z-x., and Mathews, F. S. (1990) J. Mol. Biol. 212,837-863 Laheyrie, F., Groudinsky, O., Jacquot-Armand, Y., and Naslin, L. (1966) Biochim. Biophys. Acta 128,492-503 Guiard, B., and Lederer, F. (1974) Proc. Natl. Acad. Sci. U. S. A. 7 1 , 2539-

2543 12. Volokita, M., and Somerville, C. R. (1987) J . Bid. Chem. 2 6 2 , 1582515828 13. Cederlund, E., Lindqvist, Y., Soderlund, G., Branden, G I . , and Jornvall, H. (1988) Eur. J. Biochem. 173,523-530 14. Lederer, F., Cortial, S., Becam, A. M., Haumont, P.Y., and Perez, L. (1985) Eur. J. Biochem. 139,59-65 15. Guiard, B. (1985) EMBO J. 4 , 3265-3272 16. Lim, L. W., Shamala, N., Mathews, F. S., Steenkamp, D. J., Hamlin, R., and Xuong, N. (1986) J. Biol. Chem. 2 6 1 , 15140-15146 17. Lindqvist, Y., and Brandkn, C:I. (1989) J. Biol. Chern. 2 6 4 , 3624-3628 18. Lim, L. W., Mathews, F. S., and Steenkamp, D. J. (1988) J. Biol. Chem. 263,3075-3078 19. Jones, T. A,, Bergdoll, M., and Kjeldgaard, M.(1990) in Crystallographic &

Modeling Methods in Molecular Design (Bugg, C., and Ealick, S. eds) pp.

189-199, Springer-Verlag, Berlin 20. Rossmann, M. G., and Argos, P. (1975) J. Biol. Chem. 2 5 0 , 7525-7532 21. Lebioda, L., Hatada, M. H., Tulinsky, A., and Mavridis, I. M. (1982) J. Mol. Biol. 1 6 2 , 445-458 22. Lindqvist, Y.,and Branden, C.-I. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 6855-6859 23. Ohlsson, I., Nordstrom, B., and Branden,G I . (1974) J. Mol. Biol. 8 9 , 339354 24. Rossmann, M. G., Moras, D., and Olsen, K. W. (1974) Nature 2 5 0 , 194199 25. Lederer, F., and Mathews, F. S. (1987) in Flavins and Flavoproteins (Edmondson, D. E., and MacCormick, D. B., eds) pp. 133-142, Walter de

Gruyter, Berlin

26. Lederer, F. (1990) in Chemistry andBiochemistry ojFlauoenzymes (Muller,

31.

F., ed)Vol. 11, CRC Press, in press Walsh, C. T. (1980) Acc. Chem. Res. 1 3 , 148-155 Bruice, T. C. (1980) Acc. Chem. Res. 1 3 , 256-262 Ghisla, S., and Massey, V. (1989) Eur. J. Biochem. 1 8 1 , 1-17 Duhois, J., Chapman, S. K., Mathews, F. S., Reid, G. A., and Lederer, F. (1990) Biochemistry 29,6393-6399 Porter, D. J. T., Voet, J. G., and Bright, H. J. (1973) J. Biol. Chem. 2 4 8 ,

32. 33. 34. 35. 36.

Ghisla, S., and Massey, V. (1980) J. Biol. Chem. 2 5 5 , 5688-5696 Fendrich, G., and Ghisla, S. (1982) Biochim. Biophys. Acta 7 0 2 , 242-248 Genet, R., and Lederer, F. (1990) Biochem. J. 266,301-304 Hemmerich, P., and Muller, F. (1973) Ann. N. Y. Acad. Sci. 2 1 2 , 13-26 Entsch, B., Ballou,D. P., and Massey, V. (1976) J. Biol. Chem. 251,2550-

27. 28. 29. 30.

4400-4416

2563 37. Eberlein, G., and Bruice, T. C. (1983) J. Am. Chem. SOC. 105,6685-6697 38. Tegoni, M., and Mathews, F. S. (1989) J. Biol. Chem. 2 6 3 , 19278-19281 39. Lockridge, O., Massey, V., and Sullivan, P. A. (1972) J. Biol. Chem. 247, 8097-8106 40. Bright, H. J., and Porter, D. J. T. (1975) in The Enzymes, Vol. 12, 3rd ed. (Boyer, P. D., ed) pp. 421-505, Academic Press, New York 41. Baker, A., and Tolbert, N. E. (1966) Methods Enzymol. 9 , 338-342 42. Manstein, D. J.,Pai,E. F., Schopfer, L. M., andMassey, V. (1986) Biochemistry 25,6807-6816 43. Schreuder, H. A,, Hol, W. G. J., and Drenth, J. (1990) Biochemistry 2 9 , 3101-3108 44. Bolognesi, M., Ghisla, S., and Incoccia, L. (1978) Acta Crystallogr. B 3 4 , 821-828 45. Gervais, M., and Tegoni, M.(1980) Eur. J. Biochem. 1 1 1357-367 ,