Protein engineering for the elucidation of the mechanism of electron ...

Biochemical Society Transactions

796

The protective effect on non-phosphorylated connexin molecules induced by PKC phosphorylation of nearby connexin molecules appears to be mediated by as yet unknown conformational changes. This effect may be facilitated by the tight packing of connexon channels in the bidimensional membrane lattice. It is important to notice that the spreading of the protective effect to non-phosphorylated connexin molecules may increase the efficiency of this putative control mechanism by several orders of magnitude. This may be a mechanism for the prevention of disassembly of gap junctions in certain physiological conditions. This work was supported in part by Grants to A.V. from the Consejeria de Educacion de la Comunidad de Madrid (C174/90 and 306/92), and from the Comision Interministerial de Ciencia y Tecnologia (SAF392/93). M.E. is the recipient of a predoctoral fellowship from the Secretaria de Educacion de la Comunidad de Madrid.

1 Imewenstein, W. K. (1979) Biochim. Biophys. Acta 560, 1-65 2 Spray, D. C. and Bennett. M. V. 1,. (1985) Annu. Rev. I’hysiol. 47,281-303 3 Sheridan. J. L). and Atkinson. M. M. (1985) Annu. Kev. Physiol. 47, 337-353 4 Kevel, J.-P.?Nicholson, B. J. and Yancey. S. B. (1985) Annu. Kev. Physiol. 47, 263-275

5 Caveney, S. (1985) Annu. Kev. Physiol. 47,319-335 6 Saez, J. C., Connor, J. A., Spray, 11. C. and Bennett, M. V. I,. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2708-27 12 7 Willecke, K., Hennemann, H., L)ahl, E., Jungbluth, S. and Heynkes, K. (1991) Eur. J. Cell Hiol. 56, 1-7 8 Laird, D. W., Puranam, K. I,. and Revel, J.-1’. (1991) Riochem. J. 273,67-72 9 Stein, L. S., Boonstha, J. and Burghardt, R. C. (1992) Exp. Cell Kes. 198, 1-7 10 Elvira, M., Diez, J. A., Wang, K. K. W. and Villalobo, A. (1993) J. Riol. Chem. 268,14294- 14300 11 Hertzberg, E. T. and Van Eldik, I,. J. (1987) Methods Enzymol. 139,445-454 12 1,aemmli. U. K. (1970) Nature (London) 227. 680-685 13 Lowry, 0. M.. Rosebrough, N. J., Farr, A. I,. and Kandall, K. J. (1951)J. Riol. Chem. 193.205-275 14 Wang, K. K. W., Villalobo, A. and Koufogalis, B. 1). ( 1 988) Arch. Riochem. Hiophys. 260,696-704 15 Wang, K. K. W., Roufogalis, H. L). and Villalobo, A. (1988) Arch. Riochem. Riophys. 267,317-327 16 Saez, J. C., Nairn, A. C., Czernik, A. J.. Spray, L). C., Hertzberg, E. I,., Greengard, P. and Hennett, M. V. I,. (1990) Eur. J. Hiochem. 192,263-273 17 Stauffer, K. A,, Kumar, N. M., Gilula, N. H. and Unwin, N. (1991) J. Cell Riol. 115, 141-150

Keceived 5 January 1994

Protein engineering for the elucidation of the mechanism of electron transfer in redox proteins Carlos Gomez-Moreno*§, Milagros Medina*, John K. Hurleyt, Michel A. Cusanovicht, John L. Markleyt, Hong Cheng$, Biu Xiat, Young K. Chaet and Gordon Tollint *Departamento de Bioquimica y Biologia Molecular y Celular, Universidad de Zaragoza, E-50009 Zaragoza, Spain, +Department of Biochemistry, University of Arizona, Tucson, AR 8572 I , U.S.A. and $Department of Biochemistry, University of Wisconsin, Madison, WI 53706, U.S.A.

Introduction Redox reactions in living organisms constitute the ultimate source of energy for all of their biological functions. In these processes, the electrons are taken up from reduced substrates and transferred by a mechanism that involves, in many cases, protein-protein interactions. Flavoproteins are among the most important components of these electron-transfer chains, participating as they do in photosynthesis, respiration, microsomal electron transfer, nitrogen fixation, and other biosynthetic and degradative reactions. These proteins contain a Abbreviations used: dKf, 5-deazariboflavin; FNK, ferredoxin:NADP reductase. $To whom correspondence should be addressed. +

tightly bound flavin group, which is bright yellow in colour in its oxidized state and bleaches when it becomes reduced. The reduction can take place in a single step, in which the uptake of two electrons at one time produces the hydroquinone form of the flavin, or in two sequential steps, each involving the uptake of one electron, in which case the intermediate semiquinone is formed. This characteristic change in the colour of flavoproteins, which is dependent on their redox state, makes it possible to follow their participation in enzymic reactions by simply monitoring A4s0, an increase of which is a measure of the reduction of the oxidized form. The reaction can also be followed at 600 nm, where the semiquinone form exhibits maximal absorbance. This unique ability of flavoproteins to exchange

Membrane Dynamics and Transport

either one or two electrons makes them indispensable for those reactions where electrons have to be transferred from a two-electron donating substrate (such as NAD[P]H) to a single-electron acceptor, as is the case for ferredoxin or the different types of cytochromes. Although a very large amount of information has been amassed concerning the biochemistry of these proteins, the mechanisms of electron transfer and the structural and thermodynamic requirements for these processes are still unclear. This is especially true for parameters such as the thermodynamic driving force, the distance and relative orientation of the redox centres and the role of the intervening protein matrix in the electron-transfer process. Electron-transfer between a flavoprotein and another redox protein, involves the formation of a complex between the two proteins, which involves several amino acid residues from each protein. This example of molecular recognition is remarkable because the formation of the complex allows electron transfer to occur between two rather distant groups which are very much buried in the protein structure. It is generally assumed that complexes are stabilized by the formation of electrostatic interactions between amino acid residues that bear opposite charges and are located in specific positions of each protein 1-31, although clearly other forces may participate as well. It is expected that most of the amino acids involved in the formation of the productive complex would only play a role in stabilizing the complex; however, some of them could be directly involved in the electron-transfer reaction. Several theoretical models have been proposed to explain how long-range electron-transfer reactions occur. Essentially, some of these presume a requirement for a chemical pathway formed by covalent and hydrogen bonds for electron-transfer reactions [ 4 j whereas others propose that only the distance, the free energy (dependent on the redox potential of the cofactors) and the reorganization energy are important for defining the rate and direction of electron transfer [ 51.

reduced and then binds to FNR to form a rather tight complex within which electron transfer takes place. This electron-transport chain is also thought to participate in feeding electrons to nitrogenase, which is located within specialized cells called heterocysts present in nitrogen-fixing microorganisms. Moreover, the filamentous cyanobacterium Anabaena synthesizes, under conditions of iron deficiency, a small flavoprotein (flavodoxin) which replaces ferredoxin for the purposes of both photosynthesis and nitrogen fixation [6]. The three proteins from Anabaena, namely FNR, ferredoxin and flavodoxin, have been cloned and can now be expressed in Escherichziz coli at quite high yields (7-91. Furthermore, the three-dimensional structures of all these proteins have been resolved recently at high resolution [ 10- 121. Additional information has emerged from chemical modification studies concerning the involvement of certain amino acid residues in the formation of the complex between FNR and ferredoxin or flavodoxin. In particular, an arginine residue and two lysine residues have been identified in Anabaena FNR as being responsible for its interaction with ferredoxin [ 13,141. In the case of FNR, four aspartic acid residues have been identified in Anabaena flavodoxin as being essential for the binding interaction [ 151. Chemical modification and cross-linking studies have also provided important information concerning the amino acid residues involved in the formation of the complex between spinach FNR and ferredoxin [16,17]. Using this information, as well as information from sequence matching, site-directed mutants of ferredoxin have been constructed and their ability to transfer electrons to FNR has been determined kinetically as a measure of the involvement of the mutated amino acid in the electron-transfer reaction. Laser flash photolysis has been used to measure the rate of electron transfer between the two proteins. This technique is well suited to these studies because it measures directly the rate of intermolecular electron transfer independently of other processes, such as the reduction or oxidation of FNR by the reduced or oxidized substrate, that are included in other measurements of the catalytic activity.

Electron transfer between ferredoxin and ferredoxin:NADP reductase

Kinetic studies

One such electron-transport chain is that involved in the reductive side of photosynthesis. Electrons are transferred from photosystem I to ferredoxin and then to the flavoenzyme ferredoxin:NAI>P+ reductase (FNR), where NAL)P+ is finally reduced to NADPH. Ferredoxin forms a complex with the photosynthetic membrane in order to become

The flash photolysis technique is based on the generation of a strong reductant within a very short time ( < 1 ps) through the excitation of a photosensitive molecule (usually a flavin analogue) by a pulse of laser light (see [ I ] for a description of the methodology). When a solution containing this strong reductant also contains a protein, it is then

+

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second-order rate constant for ferredoxin reduction is obtained (2.2+0.2)X 1 0 x M - ' * s - l (seeTable 1). When FNR is present in the solution, the reduced ferredoxin is reoxidized by FNR, as shown by the kinetic traces presented in Figures l(c) and l(d). In the first case, the trace records the reduction and reoxidation of ferredoxin at 507nm, which is an isosbestic point for FNR, so that the spectral changes observed correspond only to those taking place in ferredoxin, whereas the second trace follows the reduction of FNR, which forms a semiquinone upon reduction by one electron at 600 nm, where ferredoxin does not absorb. The variation of the observed rate constant as a function of FNR concentration yields a linear plot, the gradient of which corresponds to a second-order rate constant for the re-oxidation by FNR of reduced ferredoxin. At high ionic strength, this rate constant becomes independent of FNR concentration at high FNR concentrations. The saturation effect is very important because this allows the determination of a firstorder rate constant for the electron-transfer reaction between ferredoxin and FNR (klYI.= 5000 s - I for wild-type ferredoxin) concomitant with the determination of the dissociation constant for the complex (Kd= 6.7 x lo5M). These results are indicative of a minimal two-step mechanism comprising a second-order reaction, k , , followed by a first-order

reduced very rapidly. If a complex of electronexchanging proteins is present in the solution, one of them is reduced preferentially and the rate of intermolecular electron transfer can subsequently be followed spectroscopically. In these experiments, the light-sensitive molecule is the flavin analogue 5-deazariboflavin (dRf), which is reduced in the presence of EDTA to the semiquinone with a mid-point redox potential of - 650 mV (for details, see [ 181). Figure 1 shows the kinetic traces of a laser flash photolysis experiment using a dRf/EDTA solution in the absence of another electron-transfer compound. In this case, the semiquinone radical dRfH', which is rapidly induced by the laser flash, partitions disproportionately into oxidized and fully reduced flavin. When oxidized ferredoxin is present in the solution in which the strong reductant dRfH' is formed, ferredoxin is rapidly reduced, as can be monitored at 465 nm, where this protein shows maximal absorbance change upon reduction. The kinetic trace shows a rapid increase of absorption corresponding to the formation of the dRfH' radical followed by a decay which extends below the preflash baseline. This is a clear demonstration that ferredoxin reduction is taking place in this experiment. By plotting the pseudo-first order rate constant as a function of ferredoxin concentration, the

Figure I

Transient decay curves of ( u ) dRf/EDTA (Asos);( b ) dRf/EDTA containing wild-type ferredoxin (A46s); and ferredoxin with FNR (c, d, A,,,) Solutions also contained I rnM EDTA in 4 rnM phosphate buffer, pH 7 (/ = 0.0 12). dRf was IOpM in ( b ) and 40pM in (c) and (d). FNR was 5pM

0.5 I

I

I

h

2

0.0

-

3 C

t. 2

-0.5

-

1

0.5 0.0 -0.5 -

I

I

I

I

.o

I

I

1

I

f.0

1.0

I

-1

-

f

0.4 0.2 0.0 .._

-0.2

0.000

0.002

0.004

Time

(5)

I

I

I

I

I

0.000

0.002

0.004

0.006

Membrane Dynamics and Transport

T . ~ b l eI

Midpoint redox potential (€,,J and second-order rate constants ( k ) for reduction of Anabaena ferredoxin by dRf semiquinone and of FNR by reduced ferredoxin ( I = 0.0 t 2 ) WT, wild.type, n.d.. not determined.

FNR were prepared. These included several negatively charged amino acids that are supposed to form an electrostatic interaction with positively charged residues in FNR and also an aromatic amino acid residue that is present in a hydrophobic patch of ferredoxin and is also located in the region of interaction with FNR.

Ferredoxin mutants Protein

Ell2

WT D68UD69K D68K E94UE95K E95K E94K E94Q E94D F65A F651 F65W F65Y S64YlF65A

- 440 n.d. n.d. n.d. n.d. - 4 3 5 f 15 n.d. n.d. - 4 4 5 f 15 n.d. n.d. n.d. n.d.

(mv)

dRfH'

2.2f 0.2 I .4f0.2 I .3f0.2 2.5f 0.4 I .7f 0.I 2.6f 0.4 I .8 f 0. I I .5f0.2 I .2_+ 0.2 1.1 f0.I

I .2f0.2 I .5f0.2 I .6 f0. I

FNR I .2f0.I 1.1 f0.I

I .9f0.2

- 0.00007 I .2f0.I

- 0.00005

- 0.00009 I .3f0.5

- 0.00007 - 0.00022 0.9 f0.I

I .3f 0.2

- 0.00005

reaction, k,, that becomes rate-limiting at high concentration of the proteins (from 15 to l 0 0 p M of FNR), thus: k

Fd,,,, + FNR,,, Fd,,,.E"R,,, 1c- Fd,,, + FNR,,, where Fd represents ferredoxin, red. reduced, and ox. oxidized. This means that at high FNR concentrations, the rate-limiting step for the whole reaction is that of the intracomplex electron-transfer process, which is then a first-order reaction. Based on our hypothesis, described earlier, that negative charges are important in the interaction of ferredoxin with FNR, and based on previous reports from our and other laboratories, mutants containing substitutions at different positions within the hypothetical interaction interface between ferredoxin and

The rate constants for reduction of any of the mutants by the photosensitizer, dRftl' , after the laser flash were the same as for the wild-type protein, as shown in Table 1. This suggests that the redox properties of the mutants are not significantly different from those of the wild-types, as is confirmed by the similarity of the redox potentials determined for some of the most relevant mutant proteins (Table 1) and by the similarities between their u.v./visible light and c.d. spectra (results not shown). In sharp contrast, the rate of reduction of FNR by the reduced ferredoxin generated with the laser flash was reduced by four orders of magnitude in the case of the double mutant E94K/E95K as well as in the mutant E94K (Figure Z), but not in the mutant E95K, where a charge reversal has been made. E95K behaved, nevertheless, similarly to the wild-type protein. This is an indication of the crucial role of E94, but not E95, which is very close to it, in determining the rate of electron transfer from ferredoxin to FNK. The binding constant for the E94K ferredoxin mutant and FNR has been measured and shows a three-fold decrease from that of the wild-type (results not shown). This relatively small change argues that the amino acid change does not influence primarily the formation of the electrostatic complex but rather specifically alters the rate of electron transfer within a transient complex with FNR. Having obtained evidence for the requirement of E94 of ferredoxin for the exchange of electrons with FNR, it was of interest to determine whether the specific role played by this residue involved the

Figure 2

Stereoview of Anabaena PCC 7 I20 ferredoxin, showing mutated residues

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Biochemical Society Transactions

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negative charge present on glutamate, which was changed into a positive charge in E94K, or the other intrinsic properties of the glutamate residue. For this purpose, two additional more conservative mutants were prepared and their ability to exchange electrons with FNR was measured. The results shown in Table 1 indicate that glutamine (E94Q), which is essentially similar to glutamate but lacks its negative charge, does not support reaction with FNK and that aspartate (E94D) behaves similarly to glutamate. Thus, it is quite clear that a negatively charged side chain is required at position 94 in Anabaena ferredoxin for reactivity towards FNR. The same experiments were carried out with the mutants F65A and FhSI, where phenylalanine was replaced by alanine or isoleucine. The effect of these substitutions on the rate of electron transfer was also very dramatic; we observed an 60000fold decrease with respect to the wild-type ferredoxin in the reduction of FNR (Table 1). Moreover, in order to determine whether or not the aromatic character of the residue at position 65 was the critical parameter in controlling reactivity, the mutants F65W and F65Y were prepared. Again, all of these mutants reacted with dRM’ as did the wild-type, and both of these mutants showed essentially wildtype behaviour towards FNR. In addition, a mutant was constructed in which S64 was replaced by an aromatic residue at the same time that F65 was replaced by alanine, to test the possibility that an aromatic residue adjacent t o position 65 would restore wild-type reactivity. However, this double mutant reacted comparably to the wild-type with respect to dRfH’ and reacted more than four orders of magnitude slower with respect to FNR. These results clearly demonstrate that ferredoxin from Anabaena requires an aromatic amino acid at position 65 for efficient electron transfer to FNR. In this case, a histidine-aromatic electrostatic interaction could be involved in the specific interaction between the two proteins; such an interaction has been recently proposed to contribute to the stability of proteins [ 191. In conclusion, the results presented herein provide clear evidence for a high degree of localization and specificity in the interface region between ferredoxin and FNR. The precise roles of €294 and F65 in mediating electron transfer between the two proteins cannot be ascertained from the present experiments. Other mutations are required, especially in FNR. Finally, it is important to determine whether or not the ferredoxin residues that critically modulate ferredoxin/FNR electron transfer also play analogous roles in electron transfer from photosystem I to ferredoxin. Experiments to ascertain this are in progress.

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W e thank L)r. J. Sancho for his helpful comments and I’uri Hernandez for her technical support. This work was supported in part by grants from CICYT (H1091-1124C02-01 to C.G.-M.) and the ITS. National Institutes of Health (L)K 15057 to G.T. and GM 21277 to M.A.C.). 1 Tollin, G. and Hazzard, J. T . (1991) Arch. Hiochem. Hiophys. 287, 1-7 2 Roberts, V. A,, Freeman, tl. C.. Olson, A. J., Tainer, J. A. and Getzoff, E. D. ( 1 99 1) J. Hiol. Chem. 266, 13431-13441 3 Coughland, V. M. and Vickery, 1,. E. (1902) J. Hiol. Chem. 267,8932-8935 4 Herantan, L). N., Onuchic, J. N., Winkler, J. K. and Gray, H. H. (1992) Science 258, 1740- 174 1 5 Moser, C.. Keske, J. M., Warncke, K.. Farid, K. S.and Lhtton, 1’. 1,. (1992) Nature (Idondon)355,796-802 6 Kazquin, I)., I’eleato, M. I,., Gomez-Moreno, C., Fillat? M. F.,Schmitz, S. and Rohme, H. (1994) in Flavins and Flavoproteins (Yagi, K., ed.), W. de Gruyter. Berlin, in the press 7 Fillat, M. F.. Horrias, W . E.and Weisbeek, 1’. J. (1991) Hiochem. J. 280, 187- 19 I 8 Alam, J., Whitaker, K. A., Krogman, D. W. and Curtis, S. E. (1 980) J. Hacteriol. 168, 1265- 127 1 9 Fillat, M. F., I’acheco, M. C. and Gomez-Moreno, C. (1994) in Flavins and Flavoproteins (Yagi, K., ed.), W . de Gruyter. Berlin, in the press 10 Kypnieski, W . K., Hreiter, I). K., Henning, M. M., Wesemberg, G., Oh, €3. H., Markley, J. I,., Kayment. 1. and Holden, H. M. (1991) Hiochemistry 30, 41264132 11 Kao, S. T., Shaffie. F., Yu, C., Satyshur, K. A., Stockman, H. J., Markley, J. I,. and Sundaralingan, M. (1992) Protein Sci. 1, 1413-1427 12 Serra, I,., Vellieux, F., Fontecilla-Camps, J., Fey, M. and Gomez-Moreno, C. (1994) in Flavins and Flavoproteins (Yagi, K., ed.). W . de Gruyter, Herlin, in the press 13 Medina, M., Mendez, E. and Gomez-Moreno, C. ( 1 992) FEHS Lett. 298,25-28 14 Medina, M., Mendez. E. and Goniez-Moreno, C. (1992) Arch. Hiochem. Riophys. 299,28 1-280 15 Medina, M., I’eleato. M. I,., Mendez, E.and GbmezMoreno, C. (1992) Eur. J. Hiochem. 203,373-370 10 Zanetti, G.. Morelli, L l , Konchi, S., Negri, A., Aliverti, A. and Curti, H. (1988) Biochemistry 27, 3753-3759 17 Ile I’ascalis, A. K., Jelesarov, I., Ackerman, F., Kopennol, W . H.. Hirasawa, M., Knaff, I). H. and Rosshard, H. K. (1993) Protein Sci. 2, 1126-1 135 18 Hurley, J. K., Salamon, Z., Meyer, T . E.,Fitch, J. C., Cusanovich. M. A,, Markley. J. I,., Cheng, H., Xia, H., Chae, Y. K., Medina, M., Gomez-Moreno, C. and Tollin, G. (1993) Biochemistry 32,9346-9354 19 Lowenthal, R., Sancho, J. and Fersht, A. K. (1992) J. Mol. Wiol. 224,759-770

Received 5 January 1994