A central problem in metalloprotein oxidation-reduction chemistry is the elucidation of the relationship between electron-transfer rate and redox-center separation.2"4 The most direct way to address this problem is to make measurements of intramolecular electron-transfer rates between redox centers in proteins in cases where the separation distance is known. An attractive system in this regard is Ru(NH3) s(His-3 3)3+-ferricytochrome c (PFenl-RuIn), whose redox centers are separated by 15 Á (Figure
A stable complex between pentaammineruthenium(III) and histidine-33 in horse heart ferricytochrome c is formed in the reaction between aquopentaammineruthenium(II) and the protein at pH 7. HPLC of the tryptic hydrolysate of the modified protein was employed to identify the pentaammineruthenium binding site. Spectroscopic measurements show that the integrity of the native structure in the vicinity of the heme c group is maintained in the ruthenium-modified protein. Matthews has shown (2-5) that Ru(NH3)5H202+ is a good reagent for modification of histidine residues in proteins, and we have found that the reaction of Ru(NH3)5H202' with horse heart cytochrome c, followed by oxidation, produces several stable Ru(NH3)53+-ferricytochrome c derivatives. Characterization of one of these derivatives, Ru(NH3)5(His-33)3+-ferricytochrome c, is reported here. Five distinct peaks (A-E) from four separate Ru(NH3)5Cl2+/ Zn (amalgam) preparations were pooled and purified as follows: Peak A (ca. 32 mg ofcytochrome c) was rechromatographed on a column (2.3 x 32 cm) ofCM52 with 85 mM sodium phosphate buffer (pH 7.0); peak B (ca. 42 mg) was rechromatographed on CM52 with 85 mM sodium phosphate buffer (column bed, 2.3 X 26 cm); peak C (ca. 48 mg) also was eluted from a CM52 column (2.3 x 19 cm) with 85 mM sodium phosphate buffer; peaks D (ca. 24 mg) and E (ca. 43 mg) were rechromatographed on CM52 with 100 mM sodium phosphate buffer (pH 7.0). Column beds were 2.3 x 19 cm (peak D) and 2.3 X 17 cm (peak E). In all cases, a flow rate of 30 ml/hr was maintained and 5-ml fractions were collected. The absorbance ofevery other fraction was read at 410 nm.
MATERIALS AND METHODSThe fractions corresponding to each ofthe major species that eluted upon rechromatography were pooled (A'-E'). All experiments reported here are for E' (concentrated solutions that were to be used within a few days were kept at 4°C; other samples were shell-frozen in liquid nitrogen and stored at -100C) or its analogue in the Rutgers' preparation. E' was analyzed for Ru by flame atomic absorption. The Ru/heme c ratio was found to be 1:1 (± 10%).Protein Modification (at Rutgers). A 20-ml solution of Ru(NH3)5H202+ (-0
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Notes trigonal-bipyramidal sites around each phosphorus. The structure of A is consistent with , 13C, and 1 'B NMR spectra which indicate only one environment for each respective type of nucleus. However the spectra do not absolutely rule out isomer B or a mixture of isomers since five-coordinate phosphorus compounds are often stereochemically nonrigid.as well as the Raman spectrum and to Professor H. C. Kelly for his helpful comments. We gratefully acknowledge use of NMR and EPR instruments purchased under NSF Grants GP-8542 and GP-18110 and the support of this work by the Robert A. Welch Foundation under Grant E-439.Registry No. Na2[(CH30)3P.BH3]2, 57325-21-0; NaCioHs, 3481-12-7; (CH30)3P-BH3, 6867-39-6.
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