HiPIPs

Reduced HiPIP/ oxidized ferredoxin [Fe4S4] +-Fe2 + 200-600 5 Dipolsu- ... 

Fig. 2. H NMR spectra of (A) oxidized spinach Fe2S2 ferredoxin (33) (B) reduced spinach Fe2S2 ferredoxin (5f) (C) oxidized Desulfovibrio gigas Fe3S4 ferredoxin (138) (D) oxidized ectothiorhodospira halophila HiPIP iso-II (23) (E) reduced Chromatium vinosum HiPIP (14) (F) fully reduced Clostridium pasteurianum 2(Fe4S4) ferredoxin (139). Chemical shift values are in ppm.

Fig. 3. Theoretically expected cysteine Hj8 chemical shifts (ppm) for iron-sulfur proteins, together with associated temperature dependences (arrows). The arrows indicate the direction where the signals move when the temperature is rsiised. The signals Eiris-ing from systems containing nonequivEilent iron ions are labeled according to the ion to which the cysteine is bound. The case of reduced HiPIP is ansdogous to that of oxidized Fd.

Fig. 5. H NMR spectrum of oxidized Ectothiorhodospira halophila HiPIP iso-II (A) and experimental temperature dependence of the shifts of the signals (B) (18).

In addition to the standard constraints introduced previously, structural constraints obtainable from the effects of the paramagnetic center(s) on the NMR properties of the nuclei of the protein can be used (24, 103). In iron-sulfur proteins, both nuclear relaxation rates and hyperfine shifts can be employed for this purpose. The paramagnetic enhancement of nuclear relaxation rates [Eqs. (1) and (2)] depends on the sixth power of the nucleus-metal distance (note that this is analogous to the case of NOEs, where there is a dependence on the sixth power of the nucleus-nucleus distance). It is thus possible to estimate such distances from nuclear relaxation rate measurements, which can be converted into upper (and lower) distance limits. When there is more than one metal ion, the individual contributions of all metal ions must be summed up (101, 104-108). If all the metal ions are equivalent (as in reduced HiPIPs), the global paramagnetic contribution to the 7th nuclear relaxation rate is given by... 

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. 8. Display of the backbone of the NMR structure of reduced E. halophila HiPIP (100) as a tube with variable radius, proportional to the backbone RMSD of each residue. The figure was generated with the program MOLMOL (143).

High-potential iron-sulfur proteins (HiPIP) form a family of small (—6-10 kDa) soluble electron transport proteins originally only found in photo synthetic representatives of the proteobacteria (for reviews,... 

An EPR study 89) on two-dimensionally ordered membranes has shown (a) a strong association of HiPIP to the photosynthetic membrane, and (b) that part of this membrane associated HiPIP forms a... 

RCII may subsequently have been transformed into RCI by formation of the Fx cluster and eventually the capturing of a soluble 2[4Fe-4S] protein as an RC-associated subunit. These additions would have allowed electrons to leave the space of the membrane and serve for reductive processes in the dark reactions of photosynthesis. Our present knowledge concerning distribution of HiPIPs among species indicate that this electron carrier would have been invented only lately within the branch of the proteobacteria. Tbe evolutionary driving... 

Fig. 1. Iron-sulfur clusters basic building blocks. In most cases the iron is tetrahe-drally coordinated by sulfur from cysteinyl residues (and labile sulfur). Variability on coordination is allowed (see text). A, Rubredoxin type FeS4 (simplest cluster, no labile sulfur) B, plant-type ferredoxin [2Fe-2S] C, bacterial ferredoxin [3Fe-4S] D, bacterial ferredoxin and HiPIP [4Fe-4S] E, novel cluster [4Fe-2S, 20] ( hybrid cluster ).

The characteristic derivative-shaped feature at g 1.94 first observed in mitochondrial membranes has long been considered as the sole EPR fingerprint of iron-sulfur centers. The EPR spectrum exhibited by [4Fe-4S] centers generally reflects a ground state with S = I and is characterized by g values and a spectral shape similar to those displayed by [2Fe-2S] centers (Fig. 6c). Proteins containing [4Fe-4S] centers, which are sometimes called HIPIP, essentially act as electron carriers in the photoinduced cyclic electron transfer of purple bacteria (106), although they have also been discovered in nonphotosynthetic bacteria (107). Their EPR spectrum exhibits an axial shape that varies little from one protein to another with g// 2.11-2.14 and gi 2.03-2.04 (106-108), plus extra features indicative of some heterogeneous characteristics (Pig. 6d). 

The heterogeneous character of the EPR spectra given by some HIPIP is probably due to the heterogeneous location of the mixed-valence pair in the [4Fe-4S] centers, which was established in detailed NMR studies (121, 122). Since a heterogeneous location of the mixed-valence pair was also observed in the case of the [4Fe-4S] centers of Chromatium vinosum ferredoxin (123), the same phenomenon may account for the complex EPR spectra displayed by these centers in some proteins (124-126). 

The spin-lattice relaxation rate of Chromatium vinosum HIPIP was measured between 5 and 50 K (103). In comparison with the [4Fe-4S] cluster of B. stearothermophilus ferredoxin, the relaxation was found to be faster below 15 K and slower above this temperature. 

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