9346 zyxwvutsrqpo Biochemistry zyxwvu 1993, 32, 9346-9354 Amino Acid Residues in Anabaena Ferredoxin Crucial to Interaction with Ferredoxin-NADP+ Reductase: Site-Directed Mutagenesis and Laser Flash Photolysis+ J. K. Hurley,* Z. Salamon,*T. E. Meyer,* J. C. Fitch,* M. A. Cusanovich,f J. L. Markley,s H. Cheng,g B. Xia,! Y. K. Chae,s M. Medina,ll C. Gomez-Moreno,ll and G. Tollin's* Department of Biochemistry, University of Arizona, Tucson, Arizona 85721, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, and Department of Biochemistry and Molecular and Cellular Biology, University of Zaragoza, Zaragoza, Spain Received April 23, 1993; Revised Manuscript Received June zyxw 18. 1993' zyxwvutsrqponmlkjihgfedcbaZYXWVUT ABSTRACT: Ferredoxin (Fd) functions in photosynthesisto transfer electrons from photosystem I to ferredoxin- NADP+ reductase (FNR). We have made several site-directed mutants of Anabaena 7120 Fd and have used laser flash photolysis to investigate the effects of these mutations on the kinetics of reduction of oxidized Fd by deazariboflavin semiquinone (dRfI-I') and the reduction of oxidized Anabaena zyx FNR by reduced Fd. None of the mutations influenced the second-order rate constant for d R m ' reduction by more than a factor of 2, suggesting that the ability of the [2Fe-2S] cluster to participate in electron transfer was not seriously affected, In contrast, a surface charge reversal mutation, E94K, resulted in a 20 000-fold decrease in the second-order rate constant for electron transfer from Fd to FNR, whereas a similar mutation at an adjacent site, E95K, produced little or no change in reaction rate constant compared to wild-type Fd. Such a dramatic difference between contiguous surface mutations suggests a very precise surface complementarity at the protein-protein interface, Mutations introduced at F65 (F65I and F65A) also decreased the rate constant for the Fd/FNR electron transfer reaction by more than 3 orders of magnitude. Spectroscopic and thermodynamic measurements with both the E94 and F65 mutants indicated that the kinetic differences cannot be ascribed to changes in gross conformation, redox potential, or FNR binding constant but rather reflect the protein-protein interactions that control electron transfer. Several mutations at other sites in the vicinity of E94 and F65 (R42, T48, D68, and D69) resulted in little or no perturbation of the Fd/FNR interaction. Kinetic experiments with the heterocyst Fd from Anabaena 7 120, which functions in nitrogen fixation, are consistent with the above mutagenesis results and also indicate that Y98, which is also closely adjacent to E94 and F65, is not a critical residue for electron transfer to FNR. These results provide clear evidence for a high degree of localization and specificity in the interface region between the two proteins which is involved in the electron transfer process. Ferredoxins (Fds)' comprise a class of low molecular weight (6-1 4 kDa) acidic electrontransfer proteins found ubiquitously in nature. They function in photosynthetic electron transfer, nitrate reduction, and carbon and sulfur metabolism (Knaff zyxwvu & Hirasawa, 1991; Lovenberg, 1973-1977). These redox proteins contain one or more iron-sulfur [Fe-S] clusters as prosthetic groups, which can be of the [2Fe2S], [4Fe4S], or [3Fe-4S] type and have reduction potentials ranging from -600 to +200 mV (Cammack, 1984; Berg & Holm, 1982; Arnon & Buchanan, 1971). "Plant-type" ferredoxins contain a single [2Fe2S] cluster in an 11-kDa peptide chain and are found in chloroplasts and cyanobacteria where they function as the terminal electron acceptor from photosystem I, transferring electrons to ferredoxin-NADP reductase (FNR), which catalyzes the reduction of NADP+ to NADPH. The Work supported in part by grants from the National Institutes of Health (DK15057 to G.T. and GM21277 to M.A.C.)and from CAICYT (B1091-1124-(202-01 to C.G.-M.). * Corresponding author. zyxwvutsrqpon t University of Arizona. 8 University of Wisconsin. 11 University of Zaragoza. e Abstract published in Advance ACS Abstracts, August 15, 1993. Abbreviations: Fd, vegetative cell ferredoxin; HFd, heterocyst cell ferredoxin;FNR, ferredoxin-NADP reductase; dRf, 5-deazariboflavin; d R W , 5-deazariboflavin semiquinone; wt, wild type; Vi, electrostatic interaction energy; zyxwvutsrq k,, reaction rate constant at infinite ionic strength; F65A, phenylalanineat position 65 replacedwith alanine;other mutations are abbreviated in similar fashion. 0006-2960/93/0432-9346$04.00/0 ferredoxin from the cyanobacterium Anabaena strain 7 120 is a member of this group and has a reduction potential of -430 mV [Salamon and Tollin (1992) and herein]. The three- dimensional structure of this Fd has recently been solved to 2.5-A resolution (Rypniewski et al., 1991) and refined to 1.9 A, making it a good candidate for the study of structur+ function relationships in this class of proteins. Cyanobacteria which fix nitrogen (such as Anabaena) also have a ferredoxin isozyme which is the electron donor to nitrogenase and is located within specialized cells called heterocysts. In Anu- baena, the heterocyst Fd is 51% identical to the vegetative form and the three-dimensional structure has also been determined (Jacobson et al., 1992, 1993). There are some interesting substitutions of otherwise conserved residues at positions R42, E95, and Y98 (see below). Previous work from these and other laboratories has firmly established the role of electrostatics in protein-protein rec- ognition in several redox systems [cf. Tollin and Hazzard (1991) andRobertseta1. (1991)l. Earlystudies(Foustetal., 1969) described the formation of complexes between various Fds and FNR and pointed to the importance of electrostatic interactions in the functioning of these proteins. Subsequent investigations (Batie & Kamin, 1984) confirmed these findings and postulated the existence of a cluster of positive charge on FNR at the Fd binding site. Indeed, recent chemical zyxwvutsrqpo 0 1993 American Chemical Society