Transient Kinetics of Electron Transfer Reactions of Flavodoxin: Ionic Strength Dependence of Semiquinone Oxidation by Cytochrome c, Ferricyanide, and Ferric Ethylenediaminetetraacetic Acid and Computer Modeling of Reaction Complexest

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing %e dependence of a rate constant upon ionic strength can yield valuable information about electrostatic interactions which occur between reactants which are electrically charged. In the present study, we have determined the ionic strength dependence of the electron transfer reactions of flavodoxin semiquinone with oxidized cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA). Although flavodoxin and cytochrome c are not natural physiological partners, a major advantage of investigating this reaction is that the structures of both classes of proteins are known (Burnett et al., 1974; Smith et al., 1977; Swanson et al., 1977). Thus, experimental and theoretical results can be evaluated in terms of this structural information. Although the particular flavodoxin used in this work has not been studied by X-ray crystallography, it is highly homologous in critical structural regions to the clostridial flavodoxin whose structure is known (Dubourdieu & Fox, 1977), and great similarities are found among the various flavodoxins which have been investigated (Mayhew & Ludwig, 1975; Simondsen & Tollin, 1980). This will be considered in more detail below. Flavodoxin is a highly acidic protein (Simondsen & Tollin, 1980), and cytochrome c is highly basic (Margoliash & Schejter, 1966). The structural studies have shown a region of uncompensated negatively charged carboxyl groups in the vicinity of the flavin mononucleotide (FMN) prosthetic group of flavodoxin (Mayhew ' From the Departments of Biochemistry and Chemistry, University of Arizona, Tucson, Arizona 85721. Received January 5, 1982; revised manuscript received September 1,1982. Supported in part by Research Grants AM 15057 (to G.T.) and GM 21534 and GM 25664 (to F.R.S.) from the National Institutes of Health. Computations were partially supported by the University of Arizona Computer Center. Software exchange and hardware maintenance for the MMSX computer graphics systems were supported by National Institutes of Health Grant RR 00936. 4 Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511. 0006-2960/82/042 1 -6366$01.25/0 ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above. & Ludwig, 1975) and a number of positively charged lysines distributed around the heme edge of cytochrome c (Salemme, 1976). Such residues are likely candidates for involvement in formation of salt links, if electrostatic interactions are important in the orientation of these redox proteins for electron transfer. The participation of lysines has, in fact, been proposed for a number of other cytochrome c electron transfer reactions [cf. Salemme (1977) and references contained therein], and a variety of experimental studies have provided support for this [cf., for example, Speck et al. (1981) and Smith et al. (1981) and references cited therein]. So as to provide a comparison with the protein-protein system, we have also investigated the oxidation of flavodoxin semiquinone by the two smaller nonprotein reagents, ferricyanide and ferric EDTA. Previous kinetic studies (Jung & Tollin, 1981) of the oxidation of flavodoxin semiquinone by either cytochrome c or ferricyanide suggest that electron transfer occurs via an outer-sphere mechanism (Marcus, 1964), involving the solvent-exposed portion of the flavin coenzyme. This, in turn, implies that the reacting prosthetic groups are near or within van der Waals contact during the electron transfer event. As well be documented below, the observed flavodoxin reaction rates depend strongly on ionic strength, which is consistent with the importance of electrostatic interactions in the stabilization of the intermolecular electron transfer complex. Similar effects have been observed for the reactions of a variety of other electron transfer proteins with both protein [e.g., see Foust et al. (1969) and Smith et al. (1981)l and inorganic redox partners [e.g., see Wherland & Gray (1976) and Cusanovich (1978)l. In the case of redox reactions between proteins and inorganic ions, a generalized electrostatic effect is postulated in which the approach of the reacting ion is influenced by charged amino acid side chains situated near the protein's prosthetic group. Similarly, the ionic strength