Azotobacter vinelandii Ferredoxin I

Abstract

The reduction potential ( ′) of the [4Fe-4S] cluster of ferredoxin I (FdI) and related ferredoxins is ∼200 mV more negative than the corresponding clusters of ferredoxin and related ferredoxins. Previous studies have shown that these differences in ′ do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S]clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized ferredoxin) was shown to have a [4Fe-4S] cluster with a very negative ′ similar to that of FdI. The observation that the sequences and structures surrounding the very negative ′ clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S]cluster of FdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher ′ ferredoxin all led to increases in ′ for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH–S bond between the introduced Ser side chain and the Sγ atom of Cys ligand 42 and an accompanying movement of water.