Christopher J. Batie

Resonance Raman studies of Rieske-type proteins

Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster.

Structural characterization of the mononuclear iron site in Pseudomonas cepacia phthalate DB01 dioxygenase using X-ray absorption spectroscopy

Abstract Phthalate dioxygenase (PDO) from Pseudomonas cepacia contains a Rieske-like [2Fe-2S] cluster and a mononuclear non-heme Fe(II) site. The mononuclear iron can be replaced by a variety of divalent metal ions, although only Fe(II) permits catalytic activity. We used X-ray absorption spectroscopy to characterize the structural properties of the mononuclear iron site and to follow the structural changes in this site as a function both of Rieske site oxidation state and of phthalate binding. Data for the mononuclear site have been measured directly for PDO substituted with Co or Zn in the mononuclear site, and by difference for the native 3-Fe protein. The mononuclear site was modeled well by low Z-ligation (oxygen or nitrogen) and showed no evidence for high-Z ligands (e.g., sulfur). The relatively short average first shell bond lengths and the absence of significant outer shell scattering suggest that the mononuclear site has several oxygen ligands. With Zn in the mononuclear site, the average bond length (2.00 Å) suggests a 5-coordinate site under all conditions. In contrast, the Co- or Fe-containing mononuclear site appeared to be 6-coordinate and changed to 5-coordinate when substrate was bound, since the first shell bond length changed from 2.08 to 2.02 Å (Co) or 2.10 to 2.06 Å (Fe). The implications of these findings for the PDO mechanism are discussed.