Although reduced crystals prepared for X-ray crystallography rapidly become oxidized on exposure to air,2Z a slurry of crystalline reduced HiPIP in contact with the m o t h e r liquor or a solution of reduced HiPIP in 0.5 M NaCI-20 mM Tris, p H 7.3, suffers little deterioration during prolonged storage at - 20 °. Acknowledgments Much of the work reported in this chapter was performed in the laboratory of Professor M. D. Kamen, supported by grants-in-aid to him: N.I.H. GM-18528 and N.S.F. BMS-7202409. Dr. T. E. Meyer is thanked for sharing his largely unpublished observations on the isolation of HiPIPs. 27 C. W. Carter, Jr., J. Kraut, S. T. Freer, N. Xuong, R. A. Alden, and R. G. Bartsch, J. Biol. Chem. 249, 4212 (1974).

[36] Rubredoxin By W A L T E R L O V E N B E R G a n d M A R G A R E T N . W A L K E R Rubredoxins are defined as proteins that serve as electron carriers and contain one or more active centers consisting of a single iron atom with four cysteine sulfur atoms serving as the ligands. Rubredoxins have been observed in a n u m b e r o f microorganisms, but not in higher plants or animals. This type o f electron carrier was first isolated from Clostridium pasteurianum, 1 and similar proteins containing a single active site have been observed in other anaerobes. 2-5 A larger protein containing two active sites has been isolated from Pseudomonas oleovorans. 6 It is only in this latter organism that a defined biological role for rubredoxin is known. In this organism rubredoxin is the electron carrier in the tohydroxylation system. 1 W. Lovenberg and B. E. Sobel, Proc. Natl. Acad. Sci. U.S.A. 54, 193 (1965). z T. C. Stadtman, in "Non-Heme Iron Proteins Role in Energy Conversion" (A. San Pietro, ed.), p. 439. Antioch, Yellow Springs, Ohio, 1965. 3 j. LeGall and N. Dragoni, Biochem. Biophys. Res. Commun. 23, 145 (1966). 4 S. G. Mayhew and J. L. Peel, Biochem. J. 100, 80 (1966). 5 H. Bachrnayer, K. T. Yasunobu, and H. R. Whiteley, Biochem. Biophys. Res. Commun. 26, 435 (1967). 6 E. T. Lode and M. J. Coon, J. Biol. Chem. 246, 719 (1971).




Assay Method Spectrophotometric. The rubredoxin content of extracts can be monitored during isolation by the characteristic visible absorption band at 490 or 495 nm. This approach lacks specificity but is useful as preparations of rubredoxin approach purity. Rubredoxin from different sources may vary slightly in the hmax for this absorption band. For any one rubredoxin, the ratio of absorbancy of this visible band to the absorbancy of the band at 280 nm can be used as an index of purity. In the case of pure rubedoxin from C. pasteurianum the A280:A4a0 is 2.4 whereas rubredoxin from P. oleovorans exhibits an A280:A495 ratio of 3.7. 6 C y t o c h r o m e c Reduction. Although there is no specific biochemical assay for rubredoxin, a procedure devised by Peterson et al.7 was described by Lovenberg s and is presented again for convenience. This procedure is based on the reduction of cytochrome c by NADPH, spinach ferredoxin-NADP reductase, and limiting amounts of rubredoxin. In this reaction rubredoxin transfers an electron from the reduced flavin enzyme to the cytochrome c. The procedure of Peterson et al.r for this spectrophotometric assay is as follows: Reaction mixtures containing 100/~mol of Tris-chloride, pH 7.5, 1 mg of bovine serum albumin, about 20/~g of purified spinach ferredoxin-NADP reductase, 9 50 nmol of horse heart cytochrome c, and 10-40 pmol of rubredoxin are placed in cuvettes. After a preincubation of several minutes to allow equilibration with experimental temperature (usually 30°), 0.3 /zmol of NADPH is added, bringing the final volume to 1.0 ml. The rate of cytochrome c reduction (AOD 550 nm) is approximately proportional to the rubredoxin concentration under these conditions. This assay is extremely sensitive. One nanomole of C. pasteurianum rubredoxin catalyzed an increase in absorbancy at 550 nm of about 2 absorbance units per minute. The blank used is a cuvette from which NADPH has been omitted, and the control is a cuvette which contains no rubredoxin. Although the assay is extremely sensitive, it is not specific; clostridial ferredoxin also catalyzes the reduction of cytochrome c, but at about 20% the rate of rubredoxin. The assay therefore is only valid when the samples contain relatively little ferredoxin.

7j. A. Peterson, M. Kusunose, E. Kusunose, and M. J. Coon,J. Biol. Chem. 242, 4334 (1967). W. Lovenberg,this series, Vol. 24, p. 477. a M. Shin, K. Tagawa, and D. 1. Arnon,Biochem. Z. 338, 84 (1963).




Purification Procedures The procedure for the isolation of rubredoxin from C. pasteurianum was presented in detail in an earlier volume, s A description of the isolation of rubredoxin from P. oleovorans adapted from Lode and Coon 6 is summarized below. This procedure provides a better yield than that described previously. 10 Cultures of P. oleovorans were grown as described by McKenna and C o o n . 11 Cells were harvested and stored at -15 ° as a paste. Unless otherwise stated, procedures are carried out at 4 °. Step 1. One kilogram of cell paste is thawed and suspended in 1100 ml of 10 mM Tris base. This suspension is homogenized for 30 sec in a Waring blender, the pH is adjusted to 7.6 with I M Tris base, and 5 mg each of ribonuclease and deoxyribonuclease are added. Cellular disruption is completed by sonication for 2 min with the temperature maintained below 13°. After dilution to 2.5 liters with water and readjustment of the pH to 7.6 with 1 M Tris base, the suspension is centrifuged at 20,000 g for 20 min. The supernatant fraction which is still turbid is again diluted to 2.5 liters and the pH adjusted 7.6. This mixture is centrifuged as above for an additional 50 min, and the supernatant fraction is retained. Step 2. The rubredoxin in the extract obtained above is adsorbed on a 6 × 25 cm DEAE-cellulose column which has been prepared under 0.5 kg/cm 2 pressure and equilibrated with 0.1 M Tris.chloride pH 7.3 (2 liters). The column with the absorbed rubredoxin is washed successively with 1 liter of 0.20 mM Tris.chloride pH 7.3 and then with 3.5 liters of 0.1 M buffer. The rubredoxin is retained on the column and is subsequently eluted by a KC1 gradient produced by siphoning 1.5 liter of 0.1 M Tris.chloride pH 7.3 containing 0.5 M KC1 into a reservoir bottle containing an equivalent volume of the same buffer. The reddish protein is eluted at about 0.2 M KCI. Fractions of 20 ml are collected and those having absorbance at 497 nm of greater than 0.25 are pooled for the next step. Step 3. The rubredoxin solution is next chromatographed on a calcium phosphate-cellulose column. This chromatographic support is prepared by mixing 200 ml of CaPO4 gel (30 mg/ml) with 60 g of Whatman CF1 fibrous cellulose powder. The column is equilibrated with 0.1 M Tris.chloride pH 7.3, containing 0.1 M KC1. The pooled sample from the preceding step is applied to this column. The rubredoxin, which is ap-

10j. A. Peterson and M. J. Coon,J. Biol. Chem. 243, 329 (1968). 11E. J. McKennaand M. J. Coon,J. Biol. Chem. 245, 3882 (1970).




parent as a colored band near the bottom of the column, is eluted by continuing to wash the columns with the equilibration buffer. Fractions (15 ml) of the eluate with an absorbance ratio A~8o:A497 of 13 or less are pooled for the next step.

Step 4. The rubredoxin is next concentrated by adjusting the pooled fractions to 60% saturation with solid ammonium sulfate at pH 7.3. After centrifugation the precipitated proteins are dissolved in a small volume of 50 mM Tris.chloride pH 7.3. The rubredoxin-containing solution is diluted with 2 volumes of water and adsorbed to a 4 x 20 cm column of DEAE-cellulose which had been equilibrated with 0.1 M Tris-chloride pH 7.3. The column is washed with an additional 200 ml of the above buffer, then the rubredoxin is eluted by preparing a gradient in which 1 liter of 0.5 M Tris.chloride is siphoned into a reservoir of 1 liter of the equilibrating buffer. Fractions (10 ml) with an absorbance ratio (A280:A497) of less than 6.5 were combined. Step 5. The rubredoxin is precipitated from the pooled solution by ammonium sulfate fractionation between 40% and 60% saturation at pH 7.3. After centrifugation, the pellet, which was essentially pure, one-iron rubredoxin, is dissolved in 0.1 M Tris.chloride, pH 7.3, and stored in the frozen state. Properties

Rubredoxin has now been isolated and characterized from a number of anaerobic organisms. The protein from each of these organisms has a molecular weight of about 6000 and a single iron atom that serves as the redox site. These proteins all have a predominance of acidic amino acids. The amino acid sequence has been determined for several of them '2-16 (see the table). In addition to these anaerobes, P. oleovorans is the only other known source of rubredoxin-like proteins. The protein from these organisms is much larger than the anaerobic rubredoxin, and it contains two active sites. 6 The amino acid sequence of this protein has also been determined.'7 As can be seen in Fig. 1, this protein is an example of very interesting molecular evolution. Clearly the amino-terminal portion of the molecule and the carboxyl-terminal portion of the molecule each have ,2 K. F. McCarthy, Ph.D. dissertation, George Washington University (1972). ,3 M. Bruschi, Biochim. Biophys. Acta 434, 4 (1976). ,4 M. Bruschi, Biochem. Biophys. Res. Commun. 70, 615 (1976). ,5 H. Bachmayer, K. T. Yasunobu, and J. L. Peel, J. Biol. Chem. 243, 1022 (1968b). ,6 H. Bachmayer, A. M. Benson, and K. T. Yasunobu, Biochemistry 7, 986 (1968a). ,7 A. Benson, K. Tomoda, J. Change, G. Matsueda, E. T. Lode, M. J. Coon, K. T. Yasunobu, Biochem. Biophys. Res. Commun. 43, 640 (1971).





2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

a f-Met

Lys Lys Tyr Thr Cys Thr Val Cys Gly Tyr Ile Tyr Asp Pro Glu Asp Gly Asp Pro Asp Asp Gly Val Asn Pro Gly

b Met

c Met

d Met

e Met

Lys Lys Tyr Val Cys Thr Val Cys Gly Tyr Glu Tyr Asp Pro Ala Glu Gly Asp Pro Thr Asn Gly Val Lys Pro Gly

Asp Ile Tyr Val Cys Thr Val Cys Gly Tyr Glu Tyr Asp Pro Ala Lys Gly Asp Pro Asp Ser Gly lle Lys Pro Gly

Asp Lys Tyr Glu Cys Ser Ile Cys Gly Tyr lie Tyr Asp Glu Ala Glu Gly Asp -Asp Gly Asp Val Ala Ala Gly

Gly Lys Phe

Glu Cys Thr Leu Cys Gly Tyr Ile

Tyr Asp Pro Ala Leu Val Gly Pro Asp Thr Pro

Asp Gly Asp Gly






28 29

Thr Asp

Thr Ser

Thr Lys

Thr Lys








31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Lys Asp Ile Pro Asp Asp Trp Val Cys Pro Leu Cys Gly Val Gly Lys Asp Glu

Asp Asp Leu Pro Ala Asp Trp Val Cys Pro Val Cys Gly Ala Pro Lys Ser Glu

Glu Asp Leu Pro Asp Asp Trp Ala Cys Pro Val Cys Gly Ala Ser Lys Asp Ala

Ala Asp Leu Pro Ala Asp Trp Val Cys Pro Thr Cys Gly Ala Asp Lys Asp Ala

Glu Asp Val Ser Glu Asn Trp Val Cys Pro Leu Cys Gly Ala Gly Lys Glu Asp







50 51 52 53 54

Glu Glu Val Glu Glu

Glu Ala Ala

Glu Lys Gly

Val Lys Met Asp

Glu Val Tyr Glu Asp

a The rubredoxins are from the followin organisms: (a) Clostridium pasteurianum; (b)

Desulfovibrio vulgaris; (c) Desulfovibrio gigas; (d) Peptostreptococcus elsdenii; (e) Micrococcus aerogenes.

extensive homology with the anaerobic rubredoxins. The polypeptide chain between residues 53 and 119, however, does not exhibit any apparent homology. If we consider the clostridial species to be more primitive than the pseudomonad species, then it would appear that rubredoxin from the latter organisms arose by means of gene duplication with the insertion of the genetic information for the center portion of the polypeptide chain. The iron-sulfur center of the rubredoxin is of course the feature of central interest in the rubredoxin. Although our initial studies 18 suggested that the active site of rubredoxin was perhaps an iron atom held in the center of four cysteinyl sulfhydryl groups, several recent studies elo18W. Lovenberg and W. M. Williams, Biochemistry 8, 141 (1969).



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340 NONHEME METALLOPROTEINS [36] Although reduced crystals prepared for X-ray crystallography rapidly become oxidized on exposure to air,2Z a slurr...
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