Rubredoxin native proteins

Rubredoxin is an electron-transfer protein with an Fe(IlI)/Fe(lI) redox couple at -0.31 V (SCE) in water (20). Our peptide model, [Fe( Cys-Pro-Leu-Cys-OMe)2] (Z = benzyloxycarbonyl) (21) exhibits its Fe(lll)/Fe(ll) redox couple at -0.50 V (SCE) in Mc2SO (9). This is similar to the value observed for the native protein when the difference of the solvent is taken into account. When the model complex is solubilized in water by formation of micelles with addition of the non-ionic detergent, Triton X-KX), we also observed a quasi-reversible redox couple at -0.37 V (SCE) (5). The Fe(lll) complexes of Cys-X-Y-Cys peptides also exhibit a characteristic MCD band at 350 nm due to ligand-to-metal charge transfer which has also been found in oxidized rubredoxin (4). 

FIGURE 8.3 A hypothetical series of isomorphous heavy atom derivatives for a crystalline macromolecule, represented here by the polypeptide backbone of rubredoxin. (a) The apo-protein, stripped of its metal ion, provides native structure factors />, shown in vector and waveform on the right (b) the protein with its naturally bound iron atom and FHi, the first derivative structure factor (c) the protein with its iron plus an attached mercury atom, and the resultant structure factor Fm from the double derivative (d) a second multiply substituted derivative formed by attachment of a gold atom to the protein-iron complex. This last derivative is only marginally useful, however, since the reaction with gold also produces a modification in the tertiary structure of the protein (denoted by an arrow). Since this non-isomorphism is equivalent to introducing a nonnative structure factor contribution, the observed F s cannot be properly accounted for, and an erroneous heavy atom contribution / results. This final derivative will yield an inaccurate phase estimate 0v for the native protein. 

The ORD and CD spectra of 1-Fe rubredoxins from various sources have been published (211, 217, 219, 225, 234, 235). The changes in the ORD spectra of the C. pasteurianum aporubredoxin and of the iron protein (Fig. 13) in the oxidized and reduced forms where the aromatic amino acids absorb, namely from 260 to 300 nm, are suggestive that the environments around these side chain chromophores might change upon binding and reduction of the Fe3+ ion (235). In case of the M. aerogenes rubredoxin, it has been noticed (221) that the 280 nm extinction coefficient of the native protein is about twice what could be accounted for in terms of its tryptophanyl and tyrosyl contents, which more closely... 

Fe Protein. Characteristics of the Fe protein from Azotobacter 101) and Clostridia (94, 108) are summarized in Table VII. Clostridial Fe protein is stated to be 90-95% pure and the absence of tryptophan (102) suggests that almost all of the contaminating proteins have been removed. Recent preparations of Azotobacter Fe protein show specific activities even higher than those of clostridial Fe protein (101). The clostridial protein has a molecular weight of 39,000 and contains 2-3 Fe and 2 S atoms (94, 108). Molybdenum is absent, no ESR is detectable in the native protein (102) and, surprisingly, a resonance at g value of 1.94 has not been observed on reduction. All other purified iron-sulfur proteins with the exception of the atypical HIPIP and rubredoxin exhibit resonance in this area on reduction. One may suggest that the Fe protein is an atypical iron-sulfur protein or requires additional examination at 4°K for a resonance at g = 1.94. The individual protein has no activity alone but has all N2ase activities in combination with the Mo-Fe protein. 

The difference in redox potential and in thermal stability between native rubredoxin and the simple model complexes has been suggested to be brought about by the different protein environments (18). This is as yet unproved, however. The amino acid sequences of many rubredoxins isolated from various sources have been determined, as shown in Fig. 5 (19). A sequence around the Fe active site, Cys-X-Y-Cys, is an invariant fragment and primarily determines the chemical and physical properties. For example, C. pasteurianum rubredoxin has such sequences, Cys6-Thr-Val-Cys9 and Cys39-Pro-Leu-Cys42. 

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