Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

Abstract

The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by sitedirected mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of theThe field of iron-sulfur cluster biochemistry has been revitalized by the important discovery that this class of metalloprosthetic center may also play an active role in nonredox enzymatic chemistry, as exemplified by the centers in Fehydrogenase, aconitase, and endonuclease III (1-3). Extensive studies of model iron-sulfur cluster complexes, native and mutant ferredoxins, and high-potential iron proteins (HiPIPs) have greatly advanced the understanding of cluster chemistry in proteins (4-7). An important goal of these studies is elucidation of the structural and stereoelectronic factors that regulate the electrochemical potential of the cluster. To extend this work to systematic structure-function studies, we recently reported an efficient expression system for Chromatium vinosum HiPIP (8).Previously it has been suggested that the reduction potential of protein-bound [Fe4S4] centers is modulated by the hydrogen bonding pattern to the cluster (9, 10). Recently, however, the role of hydrogen bonding in modulating reduction potentials has been questioned (4, 7, 10). Rather, the number and orientations of hydrophobic residues that surround the cluster (Fig. 1), solvent accessibility, and the influence of the proteinderived electrostatic field have been suggested to play a major role (4, 7). Warshel, Stephens, and co-workers have addressed this issue by a rigorous computational approach (4, 7). Their results support the idea that neither hydrogen bonding nor neighboring ionized and protonated residues significantly influence cluster reduction potentials. Rather, Langevin dipoles from local backbone amides (not necessarily those involved in hydrogen bonding) appear to play a dominant role, while a significant influence from solvent dipoles has also been proposed (7). To address these issues we have embarked on a systematic program to evaluate the functional role of each of the hydrophobic residues surrounding the cluster core of C. vinosum HiPIP. Our initial studies have focused on a conserved residue that lies in close proximity to the reduced (Fig. 1) (11) and has been implicated in defining the cluster potential and electron-transfer pathways. Surprisingly, we find the hypothesis that Tyr-19 modulates the electronic properties of the cluster to be unfounded. In t...

Redox chemistry of the Schizosaccharomyces pombe ferredoxin electron-transfer domain and influence of Cys to Ser substitutions

“…The circular dichroism spectra have shown that Hs Fd and Sp etp Fd proteins have similar patterns of spectra in near-UV and visible regions, with only a minor difference in the range 300 nm to 440 nm, consistent with the moderate difference in E°’ for the two species. In line with prior studies where systematic substitutions of amino acid residues have been made around cluster binding sites [26, 37–39], the potential must by sensitive to subtle variations in the polarity of the cluster binding pocket as defined by local residues as well as Langevin dipoles from backbone amides [26]. The apparent greater stability of the Ser derivatives toward degradation, relative to other modified Fd s, is also consistent with a deeper burying of the cluster within the protein and a lower dielectric, which in the case of the native Fd would also tend to lower the potential to the more negative value (as observed) in line with plant-type Fd s relative to the adrenodoxin class [40].…”

Metalloproteins Containing Cytochrome, Iron–Sulfur, or Copper Redox Centers

“…715 Removal of this protection resulted in degradation of the cluster through a [3Fe–4S] intermediate as evidenced by heteronuclear multiple quantum coherence (HMQC) NMR. 875 Some HiPIPs form higher quaternary structures; HiPIP from Tb.…”

PqqE fromMethylobacterium extorquensAM1: a radicalS-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen

“…It is widely accepted that almost all radical SAM enzymes utilize the 5 0 -deoxyadenosyl radical produced by the homolytic cleavage of SAM to catalyse a variety of subsequent coupled reactions (4347 cluster in HiPIPs is resistant to oxidative damage because of its location in the protein interior shielded from the solvent (66,67). In marked contrast to the large majority of radical SAM proteins studied so far, PqqE from M.extorquens AM1 is unusually tolerant to oxygen; it can be purified even under fully aerobic conditions in a soluble form retaining significant SAM cleavage activity, as described in this article.…”