HiPIP spectroscopy

Oxidized, active Fe hydrogenase exhibits an EPR spectrum (g = 2.10,2.05, 2.01 [74]) that could come from low-spin Fec(III). MCD spectroscopy excludes the possibility of a so-called HiPIP, [4Fe-4S]3+ cluster [85,86], It thus appears that the active H cluster contains both an —Fe(CN)2CO-like moiety and a regular [Fe-4S] cubane, i.e., a total of 5Fe. 

Indeed it may be that 3Fe clusters are always extruded as [2Fe-2S] clusters, an observation that would have structural implications. The use of resonance Raman and MCD spectroscopy " has allowed valuable distinctions to be made between these clusters, while ESR techniques seem to be of particular value. Thus, the first indication of a 3Fe cluster in an iron-sulfur protein has usually been the observation of a g = 2.01 signal in the ESR spectrum, but it should be noted that there may be confusion with oxidized HiPIP clusters. 

Fluorine-labeled analogues of C. vinosum high-potential iron protein have been investigated by F NMR spectroscopy. By incorporation of specific fluorine-labeled amino acid residues, one can insert unique probes at well-defined locations within the protein core. The synthesis and purification of 2-, 3-, and 4-fluorophenylalanine (abbreviated 2-F-, 3-F-, and 4-F-Phe, respectively), 3-fluorotyrosine (3-F-Tyr), and 5-fluorotr3q)tophan (5-F-Trp) derivatives of C. vinosum HiPIP, the assignment of F NMR resonances, the measurement of longitudinal relaxation times, and the temperature dependence of F and resonances have all been reported 42, 43, 136). These measurements were used to examine structural perturbations of mutants, the dynamics of interaction of residues with the cluster, and solvent accessibility, and as a test of the relative contribution of cross-relaxation to magnetization decay. 

High potential iron-sulfur protein (HiPIP) is a special type of Fd which has been isolated from some photosynthetic bacteria and detected by ESR spectroscopy in other bacteria. HiPIP also contains a single 4Fe-4S cluster, but it differs from the other Fd in having a positive standard potential of about-h 350 mV (most Fd have standard potentials in the range of the hydrogen electrode, about -420 mV). Furthermore, the HiPIP from Chromatium is paramagnetic in the oxidized state. 

ENDOR patterns as (basic) doublets separated by about 1 MHz and centered at about 16.5 MHz (mixed-valence) and 9.25 MHz (ferric pair). Therefore, as for the [2Fe2S] clusters, there should be considerable overlap with proton resonances when the measurements are taken at the usual X-band frequencies. Fortunately, the results for E. halophila II are shown in Figure 17 for three g-factors have better resolution than the ones discussed above for the [2Fe2S] clusters. Although there is no distinction possible between the individual irons within one tine group, the mixed-valence pair and the ferric pair are nicely distinguishable from the protons when the Fe sample is used for comparison (data not shown). Comparison with the available data based mostly on MSssbauer spectroscopy from several HiPIP... 

Dilg AWE, Capozzi F, Mentler M, Iakovleva O, Luchinat C, Bertini I, Parak FG. 2001. Comparison and characterization of the [Fe4S4] centre in the wild-type and C77S mutated HiPIPs from Chromatium vinosum monitored by Mdssbauer, Fe ENDOR and EPR spectroscopies. JBiol Inorg Chem 6 232-246. 

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