Abstract
The vertebrate-type Cys(4)Fe(2)S(2) ferredoxins are a class of small acidic proteins that typically act as electron shuttles between NAD(P)H-dependent reductases and monoxygenases, particularly cytochromes P450. Nuclear magnetic resonance assignments and detailed analysis of nuclear Overhauser effects permit the direct comparison of the functional C-terminal domains of three vertebrate-type ferredoxins, the mammalian adrenodoxin (Adx) and the bacterial ferredoxins putidaredoxin (Pdx) and terpredoxin (Tdx). In particular, homologous hydrogen-bonding networks involving a conserved basic residue (His 49 in Pdx, His 56 in Adx, Arg 49 in Tdx) are detailed. This hydrogen bond network appears to play a role in the mechanical transmission of redox-dependent conformational and dynamic changes from the iron-sulfur binding loop to the C-terminal domain. Hydrogen/deuterium exchange measurements have been made in Adx as a function of oxidation state for comparison with previous studies of Pdx and Tdx. The results of these measurements highlight the importance of the conserved basic residue in the linkage between oxidation state and protein dynamics. Finally, a series of mutations have been made in the C-terminal domain of Pdx, including one, Y51F, that disrupts the proposed hydrogen-bonding network without perturbing steric and hydrophobic interactions in the functional domain. Although the mutant is considerably destabilized with respect to wild-type Pdx, relatively unperturbed chemical shifts for residues near the site of the mutation and NOEs between water and Phe 51 suggest that the network is reconstituted with a solvent water in place of the tyrosine hydroxyl group in this mutant.