AZOTOBACTER-VINELANDII FERREDOXIN-I - ASPARTATE-15 FACILITATES PROTON-TRANSFER TO THE REDUCED [3FE-4S] CLUSTER

The [3Fe-4S]+/0 cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E '0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spec tra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot b e explained either by a change in spin state of the cluster (Stephens, P. J., Jensen, G. M., Devlin, F. J., Morgan, T. V., Stout, C. D., Mar tin, A. E., and Burgess, B. K. (1991) Biochemistry 30, 3200-3209) or b y a major structural change (e.g. ligand exchange) (Stout, C. D. (1993 ) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influen ce of aspartate 15 on the pH dependence of the spectroscopic and elect rochemical properties of AvFdI by construction of a D15N mutant. Aspar tate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results sh ow that replacement of aspartate by asparagine results in an approxima tely 20-mV increase in E' 0 for the [3Fe-4S]+/0 cluster at high pH con comitant with an approximately 0.8-pH unit decrease in the pK of the r educed form. The major pH dependence of E' 0 is preserved as is the ef fect observed by MCD. These data eliminate the possibility that the MC D change is due to the presence of Asp-15 and support the conclusion t hat it originates in direct protonation of the [3Fe-4S]0 cluster, prob ably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron t ransfer followed by proton transfer (for reduction) and then proton tr ansfer followed by electron transfer (for oxidation). Ionized aspartat e 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH >5.5. Pr oton transfer reactions necessary for further reduction of the [3Fe-4S ]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge a fforded by Asp-15-Lys-84 conducts protons between the cluster and solv ent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism , suggesting that the rate of protonation or deprotonation of the [3Fe -4S]0 cluster may be important in vivo.