Nanosecond Photoreduction of Cytochrome P450cam by Channel-Specific Ru-diimine Electron Tunneling Wires

Abstract

We report the synthesis and characterization of Ru-diimine complexes designed to bind to cytochrome P450cam (CYP101). The sensitizer core has the structure [Ru(L_2)L‘]^(2+), where L‘ is a perfluorinated biphenyl bridge (F_8bp) connecting 4,4‘-dimethylbipyridine to an enzyme substrate (adamantane, F_8bp-Ad), a heme ligand (imidazole, F_8bp-Im), or F (F_9bp). The electron-transfer (ET) driving force (−ΔG°) is varied by replacing the ancillary 2,2‘-bipyridine ligands with 4,4‘,5,5‘-tetramethylbipyridine (tmRu). The four complexes all bind P450cam tightly:  Ru−F_8bp-Ad (1, K_d = 0.077 μM); Ru−F_8bp-Im (2, K_d = 3.7 μM); tmRu−F_9bp (3, K_d = 2.1 μM); and tmRu−F_8bp-Im (4, Kd = 0.48 μM). Binding is predominantly driven by hydrophobic interactions between the Ru-diimine wires and the substrate access channel. With Ru−F_8bp wires, redox reactions can be triggered on the nanosecond time scale. Ru-wire 2, which ligates the heme iron, shows a small amount of transient heme photoreduction (ca. 30%), whereas the transient photoreduction yield for 4 is 76%. Forward ET with 4 occurs in roughly 40 ns (k_f = 2.8 × 10^7 s^(-1)), and back ET (Fe^(II) → Ru^(III), k_b ≈ 1.7 × 10^8 s^(-1)) is near the coupling-limited rate (k_(max)). Direct photoreduction was not observed for 1 or 3. The large variation in ET rates among the Ru-diimine:P450 conjugates strongly supports a through-bond model of Ru−heme electronic coupling.