RATE CONSTANTS FOR INTRAMOLECULAR ELECTRON-TRANSFER REACTIONS OF RUTHENIUM-MODIFIED HISTIDINE MUTANTS OF CYTOCHROME-B(2)

The cytochrome b2 core derived from flavocytochrome b2 has been expres sed in Escherichia coli and four mutants Lys-56-->His, Lys-51-->His, A sn-42-->His and Pro-41-->His have been generated by site-directed muta genesis. Ruthenium modification of the first three mutants with [Ru(NH 3)5(H2O)]2+ led to the formation of singly modified His-56[Ru(NH3)5], His-51 [Ru(NH3)5] and His-42[Ru(NH3)5] derivatives in 30-45% yield. Ho wever no modification was observed with Pro41His. Moreover no evidence was obtained for the formation of His-19[Ru(NH3)5], where His-19 is t he unco-ordinated histidine present in wild-type and mutant cytochrome b2 forms. Characterisation of the singly modified products by inducti vely coupled plasma analyses indicates an Fe: Ru ratio of 1:1 for all three derivatives. The NMR spectra of the Ru-modified proteins reveal specific broadening by the paramagnetic Ru(III) of the characteristica lly sharp C(epsilon)H and C(delta)H resonances assigned to His-56, His -51 and His-42, while the C(epsilon)H resonance due to His-19 is unaff ected. Whereas the titration of unmodified His-56, His-51, His-42 and His-41 residues by H-1 NMR spectroscopy gave pK(a) 6.4, 6.2, 6.5 and 6 .4 respectively, His-19 did not similarly titrate in the pH 4.8-10.0 r ange, which is attributed to hydrogen bonding to a nearby residue. Usi ng pulse radiolysis to generate the methyl viologen radical, MV.+, the metastable iron(II)ruthenium(III) form of the protein was obtained. R ate constants for intramolecular electron transfer from Fe(II) to Ru(I I) were determined; 3.5 (His-56), 2.8 (His-51) and 78 s-1 (His-42) at I = 0.100 M. Edge-to-edge distances, from the nearest point on one or other of the axial haem ligands to the nearest point on the imidazole ring, are 10.8 (His-56), 9.8 (His-51) and 8.5 angstrom (His-42), and t he driving force is close to 145 mV. Using the Beratan-Onuchic approac h the most favourable pathways for electron transfer have been identif ied. For His-42 a direct through-bond pathway via axial His-43 to the haem Fe is indicated. In the case of the His-56 and His-51 derivatives , pathways that include through-space interactions appear to be domina nt.