Fepr protein

The elucidation of the crystal structures of two high-spin EPR proteins has shown that the proposals for novel Fe-S clusters are not without substance. Two, rather than one novel Fe-S cluster, were shown to be present in nitrogenase, the key enzyme in the biotic fixation of molecular nitrogen 4, 5). Thus the FeMoco-cofactor comprises two metal clusters of composition [4Fe-3S] and [lMo-3Fe-3S] bridged by three inorganic sulfur atoms, and this is some 14 A distant from the P-cluster, which is essentially two [4Fe-4S] cubane moieties sharing a corner. The elucidation of the crystal structure of the Fepr protein (6) provides the second example of a high-spin EPR protein that contains yet another unprecedented Fe-S cluster. 

This review presents an overview of the discovery of the Fepr protein, the spectroscopy that led to the suggestion that it contained a [6Fe-6S] cluster, and the subsequent crystal structure analysis that disproved this hypothesis, yet uncovered what is at a present a unique Fe-S cluster in biology. 

Fig. 1. EPR spectrum of the dithionite-reduced Fepr protein fromD. vulgaris [from (7)]. The protein was 272 ftmol dm" in 25 mmol dm Hepes buffer, pH 7.5, and was reduced under argon with 10 mmol dm sodium dithionite for 3 min at ambient temperature. EPR conditions microwave frequency, 9331 3 MHz modulation frequency, 100 kHz modulation amplitude, 0.63 mT microwave power, 200 mW temperature (relative gain) 16 K (6.3X).

Fig. 3. EPR of as-isolated Z). vulgaris Fepr protein [from (7)]. EPR conditions were the same as for Fig. 1, except for microwave power (0.32 mW), temperature (9 K), Emd relative gain (3.6X).

Subsequent studies provided a wealth of information that appeared to support the hypothesis that the Fepr protein was a genuine [6Fe-6S]-containing protein. In a biochemical study (10) the elemental analysis was meticulously repeated, and, based on an assumed molecular mass of 52 kDa, the prismane protein was found to contain 6.3 Fe atoms, averaged over as many as nine different preparations. Again, no other metals than Fe were detected, suggesting that all... 

Fig. 4. EPR redox titration of ZJ. vulgaris Fepr protein at pH 7.5 of S = J components with dithionite and ferricyanide in the presence of mediators, [from (ZZ)]. ( , ) The Fepr protein-fingerprint signal (the 3+ state) monitored at g = 1.825 (O, ) signal with aU < 2 (the 5+ state) monitored atg = 1.898 ( , ) Titration in two directions starting from the isolated protein, which corresponds approximately to the top of the bell-shaped curve. ( , O) A titration starting from the fully preoxidized state. EPR conditions microwave frequency, 9.33 GHz microwave power, 13 mW modulation amplitude, 0.63 mT temperature, 15 K.

Another major discovery was the observation of unusually high-spin EPR signals in concentrated samples of the as-isolated Fepr protein. Low-field EPR resonances were observed with g-values up to 15 (Fig. 5). This further added to the concept of a [6Fe-6S] cluster. The high-spin EPR signals could be explained only by assuming an S = f... 

Fig. 5. Effective g assignment of the low-field S = IEPR signals in D. vulgaris Fepr protein [from 11)]. The spectrum was recorded at the optimEd temperature of 12 K, that is, at which the amplitude is maximal and lifetime broadening is not significEmt. EPR conditions microwave frequency, 9.33 GHz microwave power, 80 mW modulation amplitude, 0.8 mT.

A Second Fepr Protein from Desulfovibrio desulfuricans... 

Until then, the purification of the Fepr protein had been a laborous job as a 240-L batch yielded only as little as 5 mg of protein. With the overexpression clones of the Fepr proteins, the range of proteinconsuming studies such as Mossbauer spectroscopy, EXAFS, and, last but not least, crystallization experiments was greatly extended. Thus, several groups set off to systematically investigate the spectroscopic properties of both Fepr proteins, poised at all four (proposed) redox states. 

Fig. 6. Resonance REimEin spectra of Fepr protein fromZ). vulgaris [from (28)]. Upper spectrum, Fe-enriched protein in H2 0 middle spectrum, Fe-enriched protein in H" lower spectrum, Te-enriched protein in H2" 0.

Fe-S and Fe-Fe distances in [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters are all very similar 2.3 and 2.7 A, respectively. In the [6Fe-6S] prismane model cluster, however, there is an additional Fe-Fe distance at 3.7 A (Fig. 2). If a [6Fe-6S] cluster were present in the Fepr protein, then this longer Fe-Fe distance should he visible with extended X-ray absorption fine structure (EXAFS). As a consequence, EXAFS studies were carried out at the CCLRC Synchrotron Radiation facility in Daresbury, UK. The two Fepr proteins (those of D. vulgaris and D. desulfuricans), as well as a synthetic [6Fe-6S] cluster, were subjected to an EXAFS study. Low-temperature EXAFS... 

It proved possible to crystallize the Fepr protein over a wide range of pH, 5.9-8.0, and PEG 8000 concentrations, using both sitting and hanging-drop techniques. The crystals used for the X-ray analysis were produced using the following procedure (29) ... 

Fig. 7. A typical X-ray diffraction pattern of the Fepr protein fromZJ. vulgaris (Hil-denborough). The pattern was recorded on station 9.6 at the Synchrotron Radiation Source at the CCLRC Daresbury Laboratory using a wavelength 0.87 A and a MAR-Research image-plate detector system with a crystal-to-detector distance of 220 nun. X-ray data clearly extend to a resolution of 1.5 A, or even higher. The crystal system is orthorhombic, spacegroup P2i2i2i with unit cell dimensions, a = 63.87, b = 65.01, c = 153.49 A. The unit cell contains four molecules of 60 kDa moleculEu- weight with a corresponding solvent content of approximately 48%.

X-Ray Data for the Native Fepr Protein Showing the Data Quality and Completeness to 1.71 A Resolution, A = 0.87 A... 

Full details of the structure analysis and refinement are not appropriate to this review and will be published elsewhere, but it is hoped that sufficient information has been given to validate the unusual structure of the Fepr protein from Desulfovibrio vulgaris (Hilden-borough). 

Principal Details of Refinement op Fepr Protein [RESTRAIN (50)]... 

Fig. 8. A Ramachandran plot (37) indicating the overall geometrical quality of the structure of the Fepr protein from D. vulgaris at 1.7 A resolution. Some 94% of the residues lie within the most favored regions, 5.5% in the additional allowed regions, and only one residue, N303 on the border of a disallowed region. The electron density of this residue is very well defined (see text).

C. Description of the Molecular Structure of the Fepr Protein 1. The Domain Organization of the Fepr Molecule... 

Cluster 1 is a conventional [4Fe-4S] cubane cluster bound near the N-terminus of the molecule as shown in Fig. 13. Within the cluster the Fe-S bonds range from 2.26 to 2.39 A. The cluster is linked to the protein by four cysteine residues with Fe-S distances ranging from 2.21 to 2.35 A, but the distribution of the cysteine residues along the polypeptide chain contrasts markedly with that found, for example, in the ferredoxins as indicated in Section II,B,4 [also see, for example, 41) and references therein]. In the Fepr protein all four cysteine residues (Cys 3, 6, 15, and 21) originate from the N-terminus of the molecule, and the fold of the polypeptide chain in this region is such that it wraps itself tightly around the cluster, yet keeps it near the surface of the molecule. In such a position the cluster is ideally placed to participate in one-electron transfer reactions with other molecules. 

Fig. 11. Domain 1 of the Fepr protein. The two bundles, each contEiining three heh-ces (two long and one short), are arranged almost perpendiculEir to one another.

In the Fepr protein the two clusters are some 12-13 A apart and probably within electron transfer range. However, as shown in Fig. 15, there are no obvious electron pathways involving the polypeptide chain. A tyrosine residue, Tyr 493, lies approximately midway be-... 

A second important question yet to be solved concerns the function of the Fepr protein in the organisms in which it has been found, and the mechanism by which it achieves this function. Clearly, X represents a potential substrate binding site within cluster 2, and the presence of a substrate in this position will complete the coordination of Fe5 and Fe7. The nature of X, however, remains an enigma, although... 

Mo-independent nitrogenases, 36 83-84 nitrogen fixation, 36 81-84 physicochemical properties, 36 82 Fepr protein, 47 245-247 crystallography, 47 232-233 from Desulfovibrio desulfuricans, 47 227-228, 229, 232... 

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