Electrostatic and Hydrophobic Interactions during Complex Formation and Electron Transfer in the Ferredoxin/ Ferredoxin:NADP + Reductase System from Anabaena John K. Hurley, ² Maria F. Fillat, ‡ Carlos Go ´ mez-Moreno, ‡ and Gordon Tollin* ,² Contribution from the Department of Biochemistry, UniVersity of Arizona, Tucson, Arizona 85721, and Departamento de Bioquı ´mica y Biologı ´a Molecular y Celular, UniVersidad de Zaragoza, E-50009 Zaragoza, Spain ReceiVed October 31, 1995 X Abstract: Transient kinetics and protein-protein binding measurements over a wide range of ionic strength (I) have been used to characterize the interactions occurring during complex formation and electron transfer (et) between recombinant ferredoxin (Fd) and both native and recombinant ferredoxin:NADP + reductase (FNR) from the cyanobacterium Anabaena. Between I ) 12 mM and I ) 100 mM, the dissociation constant (K d ) for the complex formed between oxidized Fd and oxidized native FNR increases from 4.5 to 8.1 μM, whereas K d for the Fd complex with recombinant FNR increases from 0.3 to 3.3 μM. For both pairs of proteins, the ionic strength dependence of k obs for the et reaction is biphasic. With native FNR, k obs increases only slightly between I ) 12 mM and I ) 100 mM, whereas for recombinant FNR k obs increases by about 4-fold over this ionic strength range. For both proteins, k obs decreases monotonically above I ) 100 mM. The dependence of k obs on FNR concentration is linear for both pairs of proteins at I ) 12 mM, with the second-order rate constant for recombinant FNR being about 3 times smaller than that for the native protein. In contrast, at I ) 100 mM the k obs values are the same for both protein pairs, and show saturation behavior with respect to the FNR concentration, indicating that et becomes rate-limiting at high FNR concentrations. Electrostatic analysis of the kinetic data above I ) 100 mM allows a prediction of the ionic strength dependence of the K d values, if electrostatic interactions are the only determinant of complex stability. The predicted dependence is dramatically larger than the observed one, indicating that hydrophobic interactions make an important contribution to complex stability. The differences in binding between native and recombinant FNR are ascribed to proteolytic cleavage at the N-terminus, which occurs during preparation of the native enzyme and which removes two positively charged residues, thereby decreasing the electrostatic interactions with Fd. The kinetic results are explained by assuming that formation of the oxidized protein-protein complex blocks the et site, and thus reaction only occurs between reduced Fd and free FNR. However, even after correction for the presence of the preexisting complex, the reactivity of FNR at I ) 12 mM is significantly lower than that at I ) 100 mM. This is ascribed to electrostatic effects which force the complex with reduced Fd to be less optimal, implying that hydrophobic interactions favor a more productive interaction between the two redox proteins. Introduction In green plant, algal, and cyanobacterial photosynthesis, ferredoxin (Fd) is the terminal electron acceptor from photo- system I. After being reduced, Fd, which is a small, acidic protein containing a single [2Fe-2S] center, interacts (in two one-electron steps) with ferredoxin:NADP + reductase (FNR), an FAD-containing flavoprotein which catalyzes the reduction of NADP + to NADPH according to eq 1, where the subscripts red and ox signify the reduced and oxidized states of the Fd, respectively. X-ray crystal structures for both Fd 1,2 and FNR 3 from Anabaena (derived respectively from the closely related strains 7120 and 7119) have been determined to high resolution, and both proteins have been cloned and overexpressed in Escherichia coli, 4,5 making them available in high quantities and making it possible to readily construct site-directed mutants. In previous work from this laboratory, 6-8 we have investi- gated the electron transfer (et) mechanisms for the reactions of wild-type and site-directed mutants of recombinant Fd from the cyanobacterium Anabaena 7120 with native FNR (natFNR) isolated from Anabaena 7119. It was shown that the observed rate constant (k obs ) for the reduction of natFNR by Fd red is linearly dependent on the concentration of FNR when the experiment is performed at low ionic strength (12 mM), but at higher salt concentrations k obs showed a nonlinear dependence on FNR concentration. More recently, following the cloning and overexpression in E. coli of the Anabaena 7119 FNR gene, 5 * To whom correspondence should be addressed. E-mail: gtollin@ ccit.arizona.edu. FAX: (520) 621-9288. ² University of Arizona. ‡ Universidad de Zaragoza. X Abstract published in AdVance ACS Abstracts, June 1, 1996. (1) Rypniewski, W. R.; Breiter, D. R.; Benning, M. M.; Wesenberg, G.; Oh, B.-H.; Markley, J. L.; Rayment, I.; Holden, H. M. Biochemistry 1991, 30, 4126-4131. (2) Holden, H. M.; Jacobson, B. L.; Hurley, J. K.; Tollin, G.; Oh, B-H.; Skjeldal, L.; Chae, Y. K.; Cheng, H.; Xia, B.; Markley, J. L. J. Bioenerg. Biomembr. 1994, 26, 67-88. (3) Serre, L.; Vellieux, F.; Fontecilla-Camps, J.; Frey, M.; Medina, M.; Go ´mez-Moreno, C. In FlaVins and FlaVoproteins 1993; Yagi, K., Ed.; Walter de Gruyter: Berlin, 1994; pp 431-434. (4) Alam, J.; Whitaker, R. A.; Krogman, D. W.; Curtis, S. E. J. Bacteriol. 1986, 168, 1265-1271. (5) Go ´mez-Moreno, C.; Martı ´nez-Ju ´lvez, M.; Fillat, M. F.; Hurley, J. K.; Tollin, G. Photosynth. Res., in press. (6) Hurley, J. K.; Salamon, Z.; Meyer, T. E.; Fitch, J. C.; Cusanovich, M. A.; Markley, J. L.; Cheng, H.; Xia, B.; Chae, Y. K.; Medina, M.; Go ´mez- Moreno, C.; Tollin, G. Biochemistry 1993, 32, 9346-9354. (7) Hurley, J. K.; Cheng, H.; Xia, B.; Markley, J. L.; Medina, M.; Go ´mez- Moreno, C.; Tollin, G. J. Am. Chem. Soc. 1993, 115, 11698-11701. (8) Hurley, J. K.; Medina, M.; Go ´mez-Moreno, C.; Tollin, G. Arch. Biochem. Biophys. 1994, 312, 480-486. 2Fd red + NADP + + H + f 2Fd ox + NADPH (1) 5526 J. Am. Chem. Soc. 1996, 118, 5526-5531 S0002-7863(95)03662-6 CCC: $12.00 © 1996 American Chemical Society