Protons HiPIP

Table III reports structural statistics relative to the solution structures of iron-sulfur proteins available from the Protein Data Bank (118). The lowest percentage of residue assignment occurs for oxidized Synechococcus elongatus Fd (119). The highest percentage of proton assignment is instead obtained for oxidized E. halophila HiPIP, with a value as high as 95% (120). A close figure was also obtained for the reduced protein (94%). In the latter case, such high values are obtained also thanks to the availability of labeled...

Fig. 6.20. Calculated temperature dependence of the hyperfine shifts of the cysteine P-CH2 protons of HiPIP II from E. halophila [63]. S 2 refers to the protons sensing the ferric pair and S34 to those sensing the mixed valence pair.

Fig. 7.3. NOE difference spectra obtained upon saturation of the hyperfine shifted signals corresponding to the P-CH2 protons of the Fe4S4-coordinated cysteines in oxidized HiPIP from C. vinosum [13]. Signals marked by x arise from saturation transfer to a small amount of reduced species [13].

Solvent accessibility of native and mutant HiPIPs has been determined by multinuclear NMR methods, in particular, by use of the H/ exchange rates of backbone amide protons, evaluated by HSQC experiments, and from the isotopic perturbation of chemical shifts of labeled native and mutant HiPIPs (43, 149). 

Fig. 9. (Top) Evaluation of midpoint potentials by spectrochemical titration experiments. Eg is the solution potential and the A values refer to the absorbances of the species. (Bottom) EXSY spectrum to evaluate electron-exchange rates. Uppercase letters denote resonances for the oxidized HiPIP, and lowercase letters indicate the reduced protein. Cross-peaks derive from the exchange of magnetization between the same proton as it switches between the oxidized and reduced protein states.

The p/ka is assigned to His-42, which through Cys-43 is directly attached to the Fe4S4 cluster. The reduction potential of HiPIP increases by 30 mV upon protonation of this residue, and the Fe-S bond lengths increase from... 

The kinetics of the oxidations of Chromatium vinosum HIPIP by several ferrocenium derivatives show no inhibitions by charged redox-inactive metal complexes, and display a pH dependence (pK = 6.90) in which protonation reduces the HIPIP reactivity by a factor of two. Electron transfer at an uncharged hydrophobic patch near Cys-46 (4 A from the Fe4S4 core to the surface), enhanced by deprotonation of His-42, is inferred from the data. The self-exchange rate constant for the HIPIPo/HIPIPr couple is estimated to be 5 x 10 s from... 

The superfamily comprises three classes cytochrome c oxidases (CcOs) , quinol oxidases, and cytochrome cbb3 oxidases CcOs are the only HCOs present in eukaryotes. Enzymes of all classes contain a binuclear heme-Cu catalytic site and all act as proton pumps. The water-soluble electron carrier, ferrocytochrome c, is the physiological electron donor for CcOs and cytochrome cbb3 oxidases. Certain CcOs can also oxidize specific high-potential iron-sulfur proteins (HiPIPs). Quinol oxidases catalyze a 2e/2H+ oxidation of various quinols to quinones as the source of electrons for O2 reduction. 

Figure 17. ENDOR spectra of Fe-enriched HiPIP of E. hal. II at three g-selections (a) g = 2.143, (b) g = 2.053, and (c) g = 2.030 (refer to EPR spectrum in Fig. 15). Two groups of Fc resonances are marked with arrows and are partly overlapping with proton lines (indicated by sticks).

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... 

Examples are drawn from reduced [2Fe-2S] clusters in ferredoxins with all-cysteine coordination, Rieske-type centers, and oxidized [4Fe-4S] clusters in HiPIP proteins. Details regarding proton and Fe hyperfme interactions are provided. These coupled with the g-tensor orientation in the molecular frame provide detailed information regarding the site of reduction or oxidation within the cluster as well as valence delocalization of the iron ions. 

Hydrogenase enzyme HiPIP Detection of Fe, Ni 3 MeV proton beam SDS-PAGE 31-33... 

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