Studies of electron-transfer reactions of redox proteins have, in recent years, attracted widespread interest and attention. Progress has been evident from both physical and biological standpoints, with the increasing availability of three-dimensional structural data for many small electron-transfer proteins prompting a variety of systematic investigations (Isied, 1985). Most recently, attention has been directed towards questions concerning the elementary transfer of electrons between spatially remote redox sites, and the nature of protein–protein interactions which, for intermolecular processes, stabilize specific precursor complexes which may be optimally juxtaposed for electron-transfer. These and other issues, including the necessary reversibility of protein interfacial interactions and the dynamic properties of proteins as carriers of electrons in biological electron-transport systems, are now being addressed in the rapidly emerging field of direct (unmediated) protein electrochemistry. It is our intention in this article to discuss developments made in this area and highlight points which we believe to have the most bearing on our current understanding of diffusion-dominated, protein-mediated electron transport at electrode surfaces. First we shall outline some basic considerations which are best considered with reference to homogeneous systems.

Ferredoxins that contain [4Fe-4S]2؉/؉ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a . Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction. 2ϩ/ϩ cluster will be present (for reviews, see Refs. 2-6). During the evolution of the 7Fe ferredoxins from the 8Fe ferredoxins, two residues were inserted into this motif between the second and third cysteines to form a CysXXCysXXXXCys motif (1). This insertion moved the second cysteine out of reach of the cluster resulting in the inability to form a [4Fe-4S] 2ϩ/ϩ cluster and the appearance of a [3Fe-4S] ϩ/0 cluster in that position (7-13). Azotobacter vinelandii ferredoxin I (The structural consequences of inserting the two additional residues between the second and third cysteines are shown in Fig. 1, which compares the structure of native AvFdI (7-9) to that of Peptococcus aerogenes ferredoxin (PaFd) (10 -13) in the [3Fe-4S] ϩ/0 cluster region of AvFdI, which has the sequence Cys 8 XXCys 11 XXXXCys 16 . The comparison in Fig. 1 reveals that the chain trace of PaFd is puckered out in AvFdI, leaving the AvFdI Cys 11 removed away from the position of the ligating cysteine in PaFd. Cys 11 could therefore not be used as a cluster ligand without substantial structural rearrangement. A further structural difference in this region is that the COOHterminal half of AvFdI, which is absent in the much smaller PaFd, wraps around the AvFdI residue 8 -16 loop shielding it from solvent (9). In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S] 2ϩ/ϩ cluster in the [3Fe-4S] ϩ/0 location of native AvFdI by providing the correct three dimensional orientation of cysteine ligands. As shown in Fig. 1, Tyr 13 of AvFdI occupies the position of the fourth ligating cysteine in PaFd so here we report the purification and characterization of Y13C FdI. EXPERIMENTAL PROCEDURESMaterials-Native AvFdI was purified as described previously (14). Ammonium sulfate was from Fisher and all other materials were obtained from the vendors listed previously (15).Mutagenesis of fdxA and Expression and Purification of Y13C FdIThe oligonucleotide used for the mutagenesis had the sequence GCAAGTACTGCCATTGTGTTGGTGCAAGTGCACCGATTG. This sequence differs from the wild-type sequence (16) by the change of codon 14 from TAC to TGC, resulting in the change of FdI residue 13 from a tyrosine to a cysteine. The success of the mutag...