Identification of arginyl residues involved in the binding of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 to its substrates

ARCHIVES

Vol.

OF RIO(‘HEMISTRY

299. No. 2. December,

AND

RIOPHYSICS

pp. 281-286,

1992

Identification of Arginyl Residues Involved in the Binding of Ferredoxin-NADP+ Reductase from Anabaena sp. PCC 7119 to Its Substrates Milagros

Medina,*

Enrique

Mendez,?

*Departamento de Bioyuimica y Biologia E-50009, Zaragoza, Spain; and TServicio

Received

April

16, 1992, and in revised

form

and Carlos Gomez-Moreno*‘l

Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, de Endocrinologia, Centro Ranch y Cajal, Madrid, Spain

August

12, 1992

Ferredoxin-NADP’ reductase from the cyanobacterium Anabaena sp. PCC 7119 was chemically modified by the a-dicarbonyl reagent phenylglyoxal. The studies of the inactivation by this compound, which is specific for arginyl residues, of both the diaphorase and NADPHcytochrome c reductase activities, characteristic of the enzyme, are indicative of the involvement of at least one group of this kind in the binding site of NADP+ and a second one implicated in the interaction with ferredoxin. After specific cleavage of a FNR sample incubated with [7-‘%]phenylglyoxal, two major labeled peptides were identified. The peptide which exhibited the higher degree of modification corresponded to residues 208-242. It contained four arginine residues but only two of them were the target of the modification: Arg224 and Arg233. Protection studies with protein substrates and sequence comparison with other reductases allow us to propose that these residues in Anabaena sp. PCC 7119 FNR must be involved in the interaction with the pyridine nucleotide. The second peptide corresponds to residues 75-103 and although it contains three arginine residues, Arg77 is the only one that exhibits the modification. This residue seems to be a key one in the interaction of this reductase with ferredoxin. 8~’ 1992 Academic Press, Inc.

The photochemical reduction of NADP’ to NADPH in isolated chloroplasts has been shown to proceed via ferredoxin and the flavoenzyme ferredoxin-NADP+ reductase (FNR,’ EC 1.18.1.2) (1). The enzyme has been ’ To whom correspondence should be addressed. Fax: 34-76-567920. “ Abbreviations used: FNR, ferredoxin-NADP+ reductase; P450R, NADPH-cytochrome P450 oxidoreductase; b,R, NADH-cytochrome b, reductase; SiR, NADPH-sulfite reductase; Fd, ferredoxin; PG, phenylglyoxal; EDC, l-ethyl[3-(3.dimethylaminopropyl)lcarbodiimide; DCPIP, dichlorophenolindophenol; TFA, t,riRuoroacetic acid; PCC, Pasteur cul0003-9861/92 $5.00 Copyright CL 1992 by Academic Press, All rights of reproduction in any form

isolated from plants (1) as well as from cyanobacteria (2, 3). The enzyme from Anabaena sp. PCC 7119 has been crystallized, the crystals have been shown to diffract up to 1.9 8, (4), and work on its structure by X-ray diffraction is in progress. The three-dimensional structure of the spinach FNR has been reported (5), and the domains of interaction with FAD and the 2’-phospho-AMP are well defined. Nevertheless, although it is proposed that the NADP’-binding site overlaps that of the 2’-phosphoAMP, they seem to be slightly shifted since NADP’ does not fit in the proposed structure. Use of chemical modification techniques has shown the presence of essential residues located in the NADP+-binding site of the FNR of different origins (6-8). Positively charged groups, such as lysine (9) and arginine (8), have also been reported to be responsible for the interaction of FNR with the electron transport protein ferredoxin. It is proposed that stabilization of the functional complexes between the two proteins, or the reductase and the pyridine nucleotide, are the result of the electrostatic interaction between their charged groups (10). It is pf interest, therefore, to determine which basic residues in FNR are responsible for the recognition of Fd and also of NADP’. There are several reports of lysine residues present in the spinach enzyme which are located either at the NADP+-binding site (6, 11, 12) or at the ferredoxin binding site (9). Lysine residues have also been recently reported to interact with NADPi and ferredoxin in the Anabaena sp. PCC 7119 enzyme (13). In a previous paper we demonstrated, nevertheless, that the integrity of at least two arginyl residues is required for maximal activity of the reductase from the cyanobacterium Anabaena variabilis; one of them is located in the NADP+-binding site and a second one is preture collection; Kobs, observed rate constant; Kd, dissociation constant. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PTH, phenylthiohydantoin. 281

Inc. reserved.

282

MEDINA,

MENDEZ,

sumably situated in the ferredoxin-binding domain (8). Those residues were not identified. We describe here the isolation and sequence analysis of the peptides which have incorporated phenylglyoxal in the Anabaena sp. PCC 7119 FNR incubation with this chemical. These peptides have been obtained upon digestion with Staphylococcus aureus protease of phenylglyoxal-modified FNR from Anabaena sp. PCC 7119. The use of Anabaena sp. PCC 7119 FNR in the present work, instead of the FNR from A. uariabilis, is due to the possibility of inducing flavodoxin synthesis in the former organism (14) but not in A. uariabilis. This allows us to have a physiological system in which to study electron transfer reactions between flavoproteins. We have determined that arginine residues of Anabaena PCC 7119 FNR situated at positions 224 and 233 are involved in the binding of the reductase with NADP+, while Arg77 is responsible for the binding to ferredoxin. MATERIALS

AND

METHODS

Materials. Anabaena sp. PCC 7119 ferredoxinNADP’ reductase, ferredoxin, and flavodoxin were purified to homogeneity as described (15). Their concentrations were determined spectrophotometrically using an extinction coefficient of 7.2 mM-’ cm-’ at 423 nm for ferredoxin, 9.4 mM-’ cm-’ at 459 nm for FNR, and 9.4 mM-’ cm-’ at 464 nm for flavodoxin. [7-i4C]Phenylglyoxal (27.6 mCi/mmol) was purchased from Amersham. Solutions of [7-14C]PG were prepared in 0.1 M sodium phosphate buffer, pH 8.0, and were diluted with nonradioactive PG to the desired specific radioactivity. Enzymatic assays. Assays for diaphorase (EC 1.8.1.4) activity with DCPIP as electron acceptor and the FNR-dependent NADPH-cytochrome c reductase (EC 1.6.99.3) activity were performed in a thermostated KONTRON Uvikon 860 spectrophotometer at 23°C to assay FNR as described (16, 17). Modification of arginine groups. All inactivation mixtures were prepared as described (8). At time intervals aliquots were withdrawn to assay the different activities. After removal of excess reagents on a small Sephadex G-25 column, control samples and treated FNR were dialyzed versus 10 mM sodium phosphate buffer, pH 7.0, or 50 mM Tris/HCl, pH 8.0. For the modification of arginine groups with [7-“C]PG, reactions were carried out as indicated above but in the presence of 5.66 mM [714C]PG (0.7 mCi/mmol), with an enzyme concentration of 4.5 KM. Noncovalent and covalent complex formation. Binding of modified FNR to ferredoxin and NADP+ was monitored by absorption difference spectroscopy (18) using a thermostated KONTRON Uvikon 860 spectrophotometer. Kd values were estimated using a program written by Duggleby (19). Covalent complex formation of FNR with ferredoxin or Aavodoxin was followed by SDSPAGE, using a 8 to 25% gradient in acrylamide in a Pharmacia Fast system, after incubation of equimolecular amounts of the proteins in the presence of EDC (5 mM) for 4 h (20). Proteins were stained with 0.1% Coomassie blue R-250 in 30% methanol and 10% acetic acid in distilled water and destained in the same solution without the dye. Isoelectrofocusing was assayed in homogeneous polyacrylamide gels covering the pH range 4 to 6.5 in a Pharmacia Fast system. Proteins were stained with 0.02% Coomassie blue R-250 in 30% methanol and 10% acetic acid in distilled water and 0.1% (w/v) CuS04. Peptide analysis. S. aweus protease strain V8 digestions were carried out at 37°C with 3:lOO (w/w) protease:FNR in 0.2 M N-methylmorpholine/acetate buffer, pH 8.2, 1% SDS overnight. Protease and undigested protein were removed by TFA precipitation. Peptides were resolved by HPLC with a KONTRON system liquid chromatograph. Reversed-phase (RP)-HPLC was performed with an Aquapore C, RP-300 7~ column

AND

GOMEZ-MORENO

(250 X 7.0 mm i.d., from Pierce). The column was eluted with acetonitrile gradients containing 0.1% trifluoroacetic acid and operated at room temperature at a flow rate of 0.7 ml/min. Radioactivity was measured by counting aliquots in Cocktail F-l Normascint (100 (~1of each 0.7.ml fraction) in a LKB 1209 RACKBETA. Peptides were sequenced in a Knauer Model 810 modular liquid phase protein sequencer equipped on line with a Knauer PTH-amino acid analyzer. The PTH-amino acids were identified and quantified on a RP-HPLC system based upon C,, column (Knauer) and gradient elution with 85% 6.5 mM sodium acetate, 15% acetonitrile, adjusted to pH 4.77 as buffer A and 100% acetonitrile as B.

RESULTS

AND

DISCUSSION

Treatment of ferredoxin-NADP+ reductase from Anabaena sp. PCC 7119 with phenylglyoxal shows behavior similar to that previously reported for the A. uariabilis strain enzyme (8). Less than 10% of either the original diaphorase or NADPH-cytochrome c reductase activities were detectable after approximately 90 min of incubation, following a pseudo-first-order kinetics reaction. The rate constants obtained for the inactivation process studied under different conditions are, in general, lower than those reported for the A. uariabilis enzyme (Table I). The rate of inactivation for the NADPH-cytochrome c reductase activity is consistently higher than that observed for the diaphorase activity, either in the absence (kobs = 6.4 vs 5.14 M-’ mini’) or in the presence of NADP+ in the inactivation mixture (kOhs= 0.59 vs 0.11 M-’ mini) as was also reported to occur with the A. uariabilis enzyme (8). These results indicate that some groups, necessary for NADPH-cytochrome c reductase activity but not for diaphorase activity, were modified during incubation in the presence of NADP+. This is further supported, in the

TABLE

I

Comparison of the Phenylglyoxal Inactivation Constants of Anabaena variabilis and Anabaena sp. PCC 7119 Ferredoxin-NADP+

Reductase

Anabaena uariabilis” Diaphorase activity +PG +PG, NADP+ NADPH-cytochrome c reductase activity +PG +PG, NADP’ +PG, Fd +PG, Fd, NADP+

Anabaena sp. PCC 7119

7.50 0.48

5.14 0.11

8.60 1.58 6.70 0.49

6.40 0.59 0.60
Inactivation mixtures contained ferredoxinNADP+ reductase phenylglyoxal (5.66 mM), and, where indicated, NADP+ (17 mM) and ferredoxin (160 pM). Other conditions: 100 mM sodium phosphate, pH 8.0, and 23°C. ’ Data from (8). Note.

(4.5

FM),

BINDING

OF Anabaena

sp. PCC 7119 FERREDOXIN-NADP+

present case, by the fact that the second substrate of the NADPH-cytochrome c reductase activity, ferredoxin, provides very effective protection against the loss of this activity (bobs = 0.6 M-1 min-‘, Table I). In fact, the rate constant for the inactivation of the Anabaena sp. PCC 7119 enzyme in the presence of ferredoxin is 10 times lower than that previously reported for the A. uariabilis FNR. This different behavior with respect to ferredoxin protection can be easily explained by the fact that some amino acid replacements have been observed to take place among the sequences of both proteins (3, 22) and these changes could produce the slightly different conformations required to increase the reactivity of the specific arginine residue which was reported to be involved in the binding to ferredoxin (8). The fact that the presence of both substrates protects completely the NADPH-cytochrome c reductase activity also supports the idea that phenylglyoxal modifies at least one of the arginyl groups required for the binding of the nucleotide and another involved in the binding to ferredoxin. As previously described, Horiike’s formula (21) allows I& calculation from inactivation experiments at different concentrations of protective agent. A Kd value of 400 PM can be calculated for the Anabaena sp. PCC 7119 FNR and NADP+ complex, which is comparable to the value of 535 PM obtained for the A. uariabilis FNR. Incubation of FNR with PG under the above-described conditions completely prevented the formation of the noncovalent complex with NADP+. These data are indicative of the involvement of arginine residues in the binding to NADP+. Nevertheless, the PG-modified enzyme forms stable complexes with ferredoxin, which are qualitatively similar to those formed with unmodified enzyme but with a 20.fold higher dissociation constant (60 vs 3.14 wM; determined in 10 mM phosphate buffer, pH 8.0). The formation of a covalent complex between the modified enzyme and ferredoxin or flavodoxin in the

FNR-Fld

FNR-Fd FNR

FNR Fld

Fd

ABCD

EFGEI

of native or treated FNRs with Aavodoxin or FIG. 1. Cross-linking ferredoxin. SDS-PAGE of reaction products after treatment of each FNR sample in the presence of Aavodoxin or ferredoxin with 5 mM EDC at room temperature in 10 mM phosphate buffer, pH 7.0, for 3 h. The ratio of FNR to the partner protein was 1:l (10 pM FNR). (A) and (I) FNR treated with phenylglyoxal, (B) and (H) FNR treated with phenylglyoxal in the presence of NADP+, (C) and (G) native FNR, (D) and (F) controls in the absence of EDC, and (E) marker proteins (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and wlactoalbumin). Samples A, B, C, and D were treated with flavodoxin and F, G, H, and I with ferredoxin.

283

REDUCTASE

0 WAVELENGTH

(rUtI)

FIG. 2. Spectrophotometric comparison of native FNR and PG modified-FNR from Anabaena sp. PCC 7119 in the visible region. Both samples of protein were dissolved in 50 mM Tris/HCl, pH 8.0.

presence of a carbodiimide was also checked. In both cases only traces of covalent complexes were detected (Fig. 1). These results can be easily explained since the accessibility of the charged groups is indispensable for covalent complex formation with ferredoxin and flavodoxin, while other weak forces, such as hydrophobic interactions, could contribute to the formation of the noncovalent complex with these electron transfer proteins. The spectra of the PG-modified FNR differs from that of the native enzyme in the visible region, indicating that the flavin environment has been modified upon binding to phenylglyoxal (Fig. 2). Isoelectric focusing of native and PG treated FNR was also carried out. The same four bands at pI 4.87,5.04, 5.2, and 5.4, which are observed in the native enzyme and are indicative of a not yet well established microheterogeneity, were found in the FNR treated with PG but at slightly lower isoelectric points (~1 4.2, 4.45, 4.57, and 4.7). The FNR treated with PG in the presence of 17 mM NADP+ also showed four bands at 4.45,4.57,4.7, and 4.87. The degree of modification has been estimated, by incorporation of radioactivity, in 2-3 arginine residues modified by molecule of protein. In order to determine which arginine residues in the amino acid sequence of the FNR from Anabaena sp. PCC 7119 had incorporated PG, FNR treated with [7-l”C)PG in the presence of NADP+ for 30 h was hydrolyzed by S. aureus protease strain V8 and the resulting peptides were separated by reverse phase HPLC using a gradient from water to acetonitrile in 0.1% TFA as described in the legend to Fig. 3. NADP+ was included in the incubation mixture of the enzyme with PG in order to provide a similar rate of modification of those residues involved in the binding of both substrates (8). Figure 3 shows the peptide map obtained under these conditions. A profile of the radioactivity associated with the collected fractions is also included. The reason why radioactivity peaks seem to spread so broadly is because radioactivity has been followed in 0.7-ml aliquots, and the continuous line has been drawn later (Fig. 3, bottom). One major and two smaller radioactive peaks were observed in this map of the enzyme modified in the presence

284

MEDINA,

220 nm (-1

CPM e-1

MENDEZ,

Acetonitrlle f-----)

0.3

0

:.,~~~ 50

100 TIME

150

200

(mm)

FIG. 3. High-perfomance liquid chromatography separation of the Staphylococc~.~ aureus protease peptides from Anabaena sp. PCC 7119 ferredoxin-NADP+ reductase treated with [7-“C]phenylglyoxal. After chemical modification of FNR (4.5 KM) with PG (5.66 mM) in the presence of NADP+ (3.5 mM), 10 nmol of sample were dialyzed against 0.2 M N-methylmorpholine/acetate buffer pH 8.2, digested with S. aurcus protease strain V8 and the resulting peptides were chromatographed. The peptides were eluted with a 200.min linear gradient from 0 to 55% acetonitrile. Elution was at a flow rate of 0.7.ml/min at room temperature. Absorbance was monitored at 230 nm and radioactivity was determined by counting 0.1 ml of each 0.7.ml fraction in F-l Normascint. The radioactivity recovered was up 40%, within the usual range when HPLC techniques are used.

of NADP+, since peak 1 corresponds to free [7-14C]PG that still remained with the protein. These peaks were purified and sequenced. The ratio of radioactivity incorporation in peptides 2~3~4is 2.5:7:0.5. The major peak bearing radioactivity (No. 3, Fig. 3) can be fixed in the published primary structure of Anabaena sp. PCC 7119 (22) between residues 208 and 242. Four arginine residues are present in this peptide (Arg217, Arg224, Arg233, and Arg239). During the sequencing process, radioactivity was found in the cycles corresponding to the analysis of the residues Arg224 and Arg233. These residues are located in the NADPH-binding domain of the protein and correspond to Arg235 and Lys244 respectively in the spinach enzyme (Fig. 4). Lys244 in the spinach enzyme has been shown to be involved in the binding of NADPH by chemical modification experiments (11) and also by site-directed mutagenesis (23). Arg224 of the Anabaena sp. PCC 7119 enzyme is conserved in all the FNRs sequences tested and also in other reductases as P450R, SIR, and b5R, which also present pyridine nucleotides as cofactors. Considerable sequence homology exists between FNR, P450R, SIR, and b5R in the region that contains this peptide, which makes this region a candidate for cofactor binding. Of the four arginines contained in this peptide, Arg224 is the only one which has been conserved in all the sequences compared, although all of them present a high degree of conservation in the

AND

GOMEZ-MORENO

FNRs. Arg233 is conserved in all the FNRs tested as a lysine residue and also in P450R, and SIR, while it is an arginine in the Anabaenu sp. PCC 7119 FNR. Obviously the role played by lysine in the rest of the enzymes can be performed by Arg233 in the Anubuenu sp. PCC 7119 FNR. Recently, a model for the three-dimensional structure of the spinach enzyme and its complex formation with 2’-AMP has been reported, showing that Arg235 and Lys244 are clearly shifted during ligand binding and are, therefore, directly involved in binding the 2’-phosphate (5). The known tertiary structure of the spinach FNR has been used to fit the Anabuenu sp. PCC 7119 FNR amino acid sequence. Figure 5 shows this computer model in which the positions of Arg224 and Arg233 are marked. These residues are in a loop situated between the third strand of the parallel p sheet core of the NADPH-binding domain and the fourth surrounding helix. They are exposed to the solvent and are in a region which presents a highly positive potential determined mainly by Arg224, Lys227, and Arg233 in the Anubuenu sp. PCC 7119 enzyme. The second peak bearing an important amount of radioactivity (No. 2, Fig. 3), was also purified and sequenced, corresponding to residues 75-100. In this region of the Anubuenu sp. PCC 7119 FNR sequence there are also three arginyl residues (Arg77, Arg85, and ArglOO). Arg77 presents the highest degree of modification in this peptide. This region is located in the FAD-binding domain of the protein. Arg77 of the Anubuenu sp. PCC 7119 FNR is conserved in all the FNRs as well as in the other reductases whose sequences have been compared (Fig. 4). This residue belongs to a region with a high degree of homology between cyanobacterium and higher plant FNRs, which is not more than 50% for the whole sequences and suggests the importance of these residues in the function of the protein. The high degree of conservation of this segment in P450R, FNR, bSR, and SIR suggests that it may have a specific functional role (24). In addition to FAD and NADPH, FNR binds specifically to ferredoxin. Our data indicate that the modification by phenylglyoxal prevents the binding of ferredoxin and chemical modification and cross-linking studies have implicated Lys85 and/or Lys88 in the spinach enzyme (which are conserved in all the FNRs and correspond to Lys69 and Lys72 in the Anubuenu sp. PCC 7119 FNR) in the binding to ferredoxin (9). Moreover, in order to confirm the involvement of Arg77 in the binding of ferredoxin a control experiment in which FNR has been incubated with [7-14C]PG in the presence of ferredoxin for 4 h was performed. After removal of ferredoxin and reagent on a Sephadex G-100 column, FNR was digested with S. uureus protease strain V8 and the resulting peptides were separated by HPLC. The amount of radioactivity incorporated in peak 2, which has been mentioned above contains Arg77, was negligible, while peaks 3 and 4 exhibited a level of radioactivity incorporation comparable to that obtained when

BINDING

AnabaenaPCC7119FNR

(68)

Spirulina sp. FNR

(64) (84)

Spinacea oleracea FNR Mesembryanthemum crystallinum FN R &urn satwm FNR

Anabaena

OF

D K N G K P t

*,( ***

FERREDOXTN-NADP’

******,(c***t****S*P~(;*~*j*"v****KR*i*,(,,7) *

ii

*

t

L L P Y I

I

D

L Y *

1 T u *

p G fj

,

H i

*

*

**

**y*****“********** ;G*A*

(78)

*

7119

******

(80)

*

PCC

K L R L Y S I A S T R H G 0 D V ? D K I

****H**** ** *H*********sA’**

*

sp,

* *

I

SV

* *

*

*

Y *

*

*

*

*

S S Y *

*

*

*

*

*

*

1% Q A E V;

,I *

'

p

;

*

S**DKGrVDlViKVYrKDT(91)

285

REDUCTASE

S L C V R 0 ****

L E Y K (105)

(10,)

YR*

I

*T(l21)

S A J **FG*S**V****KR*V*T(115)

NADPH+ytochromeP450

(444)!.

reductase NADPH-sulfitereductase NADH+ytochrome

(378)

0 A *

F P N S V HI S E V h

CA IT

V A V * *

*

E(479)

G V V R *

0(411)

b,

reductase

,

(59)

AnabaenaPCC7119 FNR Sprubna sp. FN R Sprnacea oleracea FNR Mesembryanthemum crystallinum FN R Pisum satwm FNR

(200)

I

\J v

*

Y K E E L E E : 0 0 kQQ* * * k L* [

Y Y P D N F R L T YA! E FPE***** L *

SREOKN

POGGRMY

I

ODRVAEhADOLW(248)

* * * * * 0 * * E * * K * * * * * t 1 i( i- N * * h * * (240) (21,) * * * * * F * i b, K F * A * * * * * * ;) r * V * * * * T * E K * F K * * * * ; * ,4 * 0 Y * V E * * (259) (192)

*

(207)vkk

+r*

1- F*K,j

K-*A*

E*

***,)r

A V**

(205) * * * * * i

*

(574)*

A R F

h *

I) G A L T 0 *

W Q ? Y

V *

t

K Y I( F *A*

r

*

*

**U

F*

i *,i

V *

*

*

*

A F *

*

*

*A

*

? *

V *

L K*

EK****i*

D K *

E K*

14 i Oy ***

[

*M

*

[]R[**(255)

* GY

*

E E*

*(253)

L L ii, R D K E \I*

*(616)

K -

*(540)

NADPH-cytochromeP450 reductase NADPH-sulfitereductase NADH-cytochrome reductase

(497)

*

*

R*

*

0 V *

*

*

G V L S *

NV

Z 3

L *

W *

h K V * E K I

*

V *H

*V

*

R *

0 GA

E *

F M I

RDY(235)

b, (192)*

R P *

*

*

*

RN

*

t. S A?

F I( *

W *

I

L D *APE

AWD*

G * G F \I 'X i

FIG. 4. Assignment of the modified residues in the Anabaena sp. PCC 7119 ferredoxin-NADP’ reductase sequence. Alignment of two regions sp. (25), FNR from Spinacea oleracea (26), FNR of the amino acid sequences of FNR from Anabaena sp. PCC 7119 (22), FNR from Spirulina from f’isum ,sati~!um (27), FNR from Mesembryanthemum crystallinum (28), NADPH-cytochrome P4,50 reductase from rat liver (29), NADPHsulfite reductase from Salmonelln typhimurium (30), and NADH-cytochrome h, reductase from human erythrocytes (31). The final alignments are were obtained by visual inspection. Asterisks indicate exact matches of the sequences referring to Anabaena sp. PCC 7119 FNR. Hyphens packing characters introduced to align the sequences. The residues in boldcase are those which, according to the present paper, are involved in the binding to NADP’ or ferredoxin. The peptides sequenced are underlined.

the modification was carried out in the absence of ferredoxin. We can conclude, therefore, that Arg77 is a key residue in the binding to ferredoxin. This residue is part of a surface loop which also presents a positive potential and is close to the exposed dimethylbenzyl portion of the flavin. In fact, the side chain of the homologous residue in the spinach enzyme, Arg93, has been reported to stabilize, by means of hydrogen bonds, the pyrophosphate group of the FAD prosthetic group (5). The proximity of the incorporated phenylglyoxal molecule to the FAD cofactor could account for the spectral perturbation that the modified protein exhibits in the visible region. No incorporation of PG has been detected in Arg85 but some traces could remain in ArglOO. Finally peak 4 corresponds to a different cleavage of peak 2.

The present work shows the importance of Arg77, Arg224, and Arg233 from the Anabaena sp. PCC 7119 in the interaction with its substrates. Since most of these arginine residues are conserved in other reductases or represent conservative substitutions (for lysine residues), we can conclude that, in these reductases, arginine residues play essential roles as basic residues in the interaction with negatively charged substrates. ACKNOWLEDGMENTS We thank Dr. M. Frey and L. Serre from the C.E.N.G., Grenoble, France, for performing the computer graphics of FNR and also Fernando Soriano for his technical assistance in the peptide sequencing. This work was supported by Grant 0397.E from the Commission of European Communities (HAP Program) and by Grant BIO 88/0413 from Comisibn Interministerial de Ciencia y Tecnologia, Spain.

REFERENCES 1. Shin,

M., Tagawa,

K., and Amon,

D. I. (1963)

Biochem. Z. 338,

84-96. 2. Susor, R 77

W. A., and Krogmann,

D. W. (1966)

3. Sancho, ,J., Peleato, M. L., G6mez-Moreno, D. E. (1988) Arch. Biochem. Biophys. 260,

FIG.5.

Stereodiagrams showing the position of the residues involved in the interaction of FNR from Anabaena sp. PCC 7119 with its substrates. In order to obtain this model, the Anabaena sp. PCC 7119 FNR sequence has been laid over the spinach tertiary structure. The most relevant Arg (R) residues are shown.

Biochim. Hiophys. Acta

120,6.5-72.

4. Serre, L., Medina, M., Gbmez-Moreno, and Frey, M. (1991) J. Mol. Rid. 218, ,5. Karplus, 60-66.

P. A., Daniels,

6. Aliverti,

A., Gadda,

.J., and Herriot,

G., Ronchi,

Hiochem. 198. 21-24.

C., and Edmondson,

200-207.

C., Fontecilla-Camps,

,J.,

271-272. J. R. (1991)

S., and Zanetti,

Science

G. (1991)

251, Eur.

J.

286

MEDINA,

7. Bookjans,

G., and Boger, P. (1979) Arch. B&hem.

MENDEZ, Biophys.

194,

3877393. 8. Sancho, J., Medina, M., and Gomez-Moreno, Biochem. 187,39-48.

C. (1990) Eur. J.

9. Zanetti, G., Morelli, D., Ronchi, S., Negri, A., Aliverti, B. (1988) Biochemistry 27, 3753-3759.

A., and Curti,

10. Knaff, D. B., and Hirasawa, M. (1991) Biochim. Biophys. Acta 1056,

93-125. 11. Chan, R. L., Carrillo, N., and Vallejos, R. H. (1985) Arch. Biochem. Biophys. 240, 172-177. 12. Apley, E. C., and Wagner, R. (1988) Biochim. Biophys. Acta 936, 269-279. 13. Medina, M., Mendez, E., and Gomez-Moreno,

M. (1992) FEBS I.&.

298,25-28. 14. Fillat, M. F., Sandmann, G., and Gomez-Moreno, Microbial. 150, 160-164. 15. Pueyo, J. J., and Gomez-Moreno,

C. (1988) Arch.

C. (1991) Prep. Biochem. 21,191-

204. 16. Avron,

M., and Jagendorf,

A. T. (1956) Arch. Biochem. Biophys.

65,475-490. 17. Shin, M. (1971) Methods Enzymol. 23, 440-442. 18. Foust, G. P., Mayhew,

S. G., and Massey, V. (1969) J. Biol. Chem.

244,964-970. 19. Duggleby, R. G. (1981) Anal. Biochem. 110,9-18.

AND

GOMEZ-MORENO

20. Pueyo, J. J., Sancho, J., Edmondson, D. E., and Gomez-Moreno, M. (1989) Eur. J. Biochem. 183, 539-544. 21. Horiike, K., and McCormick, D. B. (1980) J. Theor. Biol. 84, 691L

708. 22. Fillat, M. F., Bakker, H. A. C., and Weisbeek, P. J. (1990) Nucleic Acids Res. 18, 7161. 23. Aliverti, A., Lubberstedt, T., Zanetti, G., Herrmann, L. G., and Curti, B. (1991) J. Biol. Chem. 266, 17760-17763. 24. Porter, T. D., and Kasper, C. B. (1986) Biochemistry 25, 1682-

1687. 25. Yao, Y., Tamura, T., Wada, K., Matsubara, H., and Kodo, K. (1984) J. Biochem. 95, 1513-1516. 26. Karplus, P. A., Walsh, K. A., and Herriott,

J. R. (1984) Biochemistry

23,6576-6583. 27. Newman, B. J., and Gray, J. C. (1988) Plant Mol. Biol. 10, 511520. 28. Michalowski, C. B., Schmitt, J. M., and Bohnert, H. J. (1989) Plant Physiol. 89, 818-822. 29. Porter, T. D., and Kasper, C. B. (1985) Proc. N&l. Acad. Sci. USA

82,9X-971. 30. Ostrowski, J., Barber, M. J., Tueger, D. C., Miller,

B. E., Siegel, L. M., and Kredich, N. M. (1989) J. Biol. Chem. 264,15796-15808. 31. Yubisui, T., Miyata, T., Iwanaga, S., Tamura, M., Yoshida, S., Takeshita, M., and Nakajima, H. (1984) J. Biochem. (Tokyo) 96, 579-

582.