Circular dichroism and magnetic circular dichroism of iron-sulfur proteins

Abstract

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for the 2-Fe ferredoxins from Pseudomonas putida and Spirulina maxima, Chromatium HIPIP, the 4-Fe ferredoxin from Bacillus stearothermophilus, and the 8-Fe ferredoxin from Clostridium pasteurianum. The spectral range spans the near-infrared, visible, and near ultraviolet. In all cases except oxidized 2-Fe ferredoxins, electronic absorption is observed continuously from less than 5000 cm-1 to above 30,000 cm-1. The CD spectra of the two 2-Fe ferredoxins are similar. In contrast, the CD of the 4-Fe and 8-Fe proteins, for a given 4-Fe cluster oxidation level, varies considerable with protein. MCD is less sensitive to protein environment than is CD. In the 2-Fe proteins, MCD at 5 T is appreciably smaller than the CD; in the 4-Fe and 8-Fe proteins, MCD and CD are comparable in magnitude. Both CD and MCD are more highly structured than the corresponding absorption spectra. The CD and MCD spectra reported provide a broader base than heretofore available for the characterization of iron-sulfur proteins containing 2-Fe and 4-Fe clusters and for the evaluation of electronic structural models for these clusters.

The Two [4Fe-4S] Clusters in Chromatium vinosumFerredoxin Have Largely Different Reduction Potentials

“…2. These data qualitatively confirm and extend previous reports (11,20). The spectra of the fully oxidized protein with [4Fe-4S] 2ϩ clusters contain several moderately intense features all over the spectral range shown.…”

“…The prominent CD bands at 366 (negative) and 410 nm (positive) readily disappear during the first transition. The spectral features corresponding to the second transition at lower potential are less intense and compare with the intensity observed for ferredoxins having a single [4Fe-4S] cluster (11). A likely interpretation is that the interaction between the two [4Fe-4S] clusters, the closest metal atoms of which are less than 9 Å apart (2), contributes to the …”

“…The absorption spectra are generally broad as they result from overlapping charge transfer bands in the visible-near UV range. The resolution of these spectra can be significantly improved by the implementation of circular dichroism (CD) which provides both a characteristic pattern for each cluster type and a sensitive monitor of changes affecting either the cluster or the protein (11). The use of the method as an accurate analytical tool is broadened when the CD spectra of electron transfer proteins can be monitored as a function of the applied potential (12).…”

Mechanism of Oxygen Sensing by the Bacterial Transcription Factor Fumarate-Nitrate Reduction (FNR)

“…In the absence of O 2 , the FNR CD spectrum displayed weak bands in the region 280 -800 nm with three positive features at max 330, 380, and 420 nm. The ⌬⑀ values were of the same order of magnitude as those of other proteins containing [4Fe-4S] cluster types (25,26 and ϳ84% complete at a ratio of 1.0, in agreement with the observations made by optical spectroscopy (see above). The CD spectra of FNR, the first to be reported, provide a useful means of monitoring the status of the FNR iron-sulfur cluster.…”

The pH dependent spectral properties of Clostridium pasteurianum 2[Fe4S4] ferredoxin

“…Deprotonation of such a bulky group as tyrosine is believed to bring about significant conformational variations of the protein around the cluster; we therefore decided to monitor the high pH transition of CpFd by CD spectroscopy, a technique particularly sensitive to conformational rearrangements at the active site of metalloproteins. As reported, the visible CD spectrum of oxidized CpFd is characterized by two intense positive bands, respectively, located at 410 and 565 nm, plus a broad negative band around 700 nm [22,23]. The spectrum is pH independent from pH 7 to 9 (see also [14]).…”