The cytochrome b₂ core derived from flavocytochrome b₂ has been expressed in Escherichia coli and four mutants Lys-56→His, Lys-51→His, Asn-42→His and Pro-41→His have been generated by site-directed mutagenesis. Ruthenium modification of the first three mutants with [Ru(NH₃)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. However no modification was observed with Pro41His. Moreover no evidence was obtained for the formation of His-19[Ru(NH₃)₅], where His-19 is the unco-ordinated histidine present in wild-type and mutant cytochrome b₂ forms. Characterisation of the singly modified products by inductively 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 characteristically sharp C^εH and C^δH resonances assigned to His-56, His-51 and His-42, while the C^εH resonance due to His-19 is unaffected. Whereas the titration of unmodified His-56, His-51, His-42 and His-41 residues by ¹H 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 range, which is attributed to hydrogen bonding to a nearby residue. Using pulse radiolysis to generate the methyl viologen radical, MV˙⁺, the metastable iron(II)ruthenium(III) form of the protein was obtained. Rate constants for intramolecular electron transfer from Fe^II to Ru^III 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 Å(His-42), and the driving force is close to 145 mV. Using the Beratan–Onuchic approach the most favourable pathways for electron transfer have been identified. 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 dominant.