IRON-SULFUR CLUSTER CYSTEINE-TO-SERINE MUTANTS OF ANABAENA [2FE-2S] FERREDOXIN EXHIBIT UNEXPECTED REDOX PROPERTIES AND ARE COMPETENT IN ELECTRON-TRANSFER TO FERREDOXIN-NADP(+) REDUCTASE

The reduction potentials and the rate constants for electron transfer let) to ferredoxin:NADP(+) reductase (FNR) are reported for site-direc ted mutants of the [2Fe-2S] vegetative cell ferredoxin (Fd) from Anaba ena PCC 7120, each of which has a cluster ligating cysteine residue mu tated to serine (C41S, C46S, and C49S). The X-ray crystal structure of the C49S mutant has also been determined. The UV-visible optical and CD spectra of the mutants differ from each other and from wild-type (w t) Fd. This is a consequence of oxygen replacing one of the ligating c ysteine sulfur atoms, thus altering the ligand --> Fe charge transfer transition energies and the chiro-optical properties of the chromophor e, Each mutant is able to rapidly accept an electron from deazaribofla vin semiquinone (dRfH(.)) and to transfer an electron from its reduced form to oxidized FNR although all are somewhat less reactive (30-50%) toward FNR and are appreciably less stable in solution than is wt Fd. Whereas the reduction potential of C46S (-381 mV) is not significantl y altered from that of wt Fd (-384 mV), the potential of the C49S muta nt (-329 mV) is shifted positively by 55 mV, demonstrating that the cl uster potential is sensitive to mutations made at the ferric iron in r educed [2Fe-2S] Fds with localized valences. Despite the decrease in t hermodynamic driving force for et from C49S to FNR, the et rate consta nt is similar to that measured for C46S. Thus, the et reactivity of th e mutants does not correlate with altered reduction potentials. The et rate constants of the mutants also do not correlate with the apparent binding constants of the intermediate (Fd(red):FNRox) complexes or wi th the ability of the prosthetic group to be reduced by dRfH(.). Furth ermore, the X-ray crystal structure of the C49S mutant is virtually id entical to that of wt Fd. We conclude from these data that cysteine su lfur d-orbitals are not essential for et into or out of the iron atoms of the cluster and that the decreased et reactivity of these Fd mutan ts toward FNR may be due to small changes in the mutual orientation of the proteins within the intermediate complex and/or alterations in th e electronic structure of the [2Fe-2S] cluster.