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

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

Quantitative imaging of cis-regulatory reporters in living embryos

A confocal laser scanning microscopy method has been developed for the quantitation of green fluorescent protein (GFP) as a reporter of gene activity in living three-dimensional structures such as sea urchin and starfish embryos. This method is between 2 and 50 times more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo. By using coinjected Texas red dextran as an internal fluorescent standard, the observed GFP intensity is corrected for variations in laser excitation and fluorescence collection efficiency. To relate the recorded image intensity to the number of GFP molecules, the embryos were lysed gently, and a fluorometric analysis of their contents was performed. Confocal laser scanning microscopy data collection from a single sea urchin blastula required less than 2 min, thereby allowing gene expression in dozens of embryos to be monitored in parallel with high spatial and temporal resolution

Enantiomeric discrimination of Ru-substrates by cytochrome P450_(cam)

Molecules with photosensitizers attached to substrates (Wilker et al., Angew. Chem. Int. Ed. 38 (1999) 90–92) or cofactors (Hamachi et al., J. Am. Chem. Soc. 121 (1999) 5500–5506) can rapidly deliver redox equivalents to buried active sites. The structure of cytochrome P450_(cam) (P450) co-crystallized with a prototypal sensitizer-substrate, [Ru-C_9-Ad]Cl_2, has been determined (Dmochowski et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12987–12990); and, in separate UV–vis absorption and time-resolved luminescence experiments, the binding of the Λ and Δ enantiomers of Ru-C_9-Ad to P450 has been measured. The results, K_D(Δ/Λ)∼2, indicate that the bipyridyl ligands of the Λ isomer interact more favorably with hydrophobic residues at the entrance to the substrate channel. We conclude that enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates