Laser Flash Spectroscopy of Zinc/Ruthenium Myoglobins: An Investigation of Distance and Medium Effects on Photoinduced Long-Range Intraprotein Electron Transfers

• Main Author: Axup, Andrew William
• Format: Thesis
• Language: English
• Published: California Institute of Technology 1987
• Subjects: Chemistry; Sperm whale
• Online Access: https://dx.doi.org/10.7907/1a3m-hb31; https://resolver.caltech.edu/CaltechTHESIS:04052019-103258317

An experimental investigation of the distance dependence of long-range intramolecular electron transfer in ruthenium-modified zinc myoglobins has been performed. The zinc/ruthenium-modified metalloproteins were prepared by substitution of zinc-mesoporphyrin IX diacid (ZnP) into four previously characterized pentaammineruthenium(III) (a5Ru) derivatives of sperm whale myoglobin (Mb). The derivatives are a5Ru(His-48)Mb, a5Ru(His-12)Mb, a5Ru(His-116)Mb, and a5Ru(His-81)Mb. Pulsed laser excitation of the zinc myoglobin produces the long-lived and highly reducing triplet excited state (3ZnP*). Electron transfer from this triplet to the ruthenium, 3znP*-Ru3+ → ZnP+-Ru2+ (ΔE° ≅ 0.8 V), was measured by time-resolved transient absorption techniques. The observed electron-transfer rates are 7.0 x 104, 100, 89, and 85 s-1 for the His-48, -12, -116, and -81 derivatives, respectively, at 25°C The electron-transfer distances were evaluated using computer modelling in which rotation about the Cα-Cβ bond of the imidazole side chain in the ruthenium-modified histidines is restricted by nonbonded repulsions with atoms at the protein surface. Recent crystallographic results for a5Ru(His-48)Mb indicate that the histidine has considerable rotational flexibility. The estimated accessible distances, both heme edge to inner coordination sphere ligand (e-e) and metal-to-metal (m-m), are as follows. For the His-48 derivative, the e-e range is 13.4-16.6 Å and the m-m range is 18.6-24.1 Å; His-12 ranges are 22.1-22.4 Å and 28.8-30.4 Å; His-116 ranges are 18.9-20.4 Å and 23.1-27.8 Å; and His-81 ranges are 19.0-19.4 Å and 26.3-26.9 Å. In addition, the orientation angles (θ) of the electron-transfer pathways with relation to the heme plane at a position of closest approach are 25° (His-48), 20° (His-12), 35° (His-116), and 25° (His-81). Fitting the rate data to an exponential distance dependence yields the expression ket = 8 x 109 exp(-β(R-4)) s-1, where β = 1.2 Å-1 and R - 4 ≥ 0 (R is the minimum e-e distance in Å). The electron-transfer rate in a5Ru(His-12)Mb(ZnP) is anomalously high (100 vs. 2 s-1 predicted by the rate-distance equation), thereby indicating that the 3ZnP*-Ru3+ electronic coupling may be enhanced by an intervening tryptophan residue that lies parallel-planar to the heme along the reaction pathway. Activation enthalpies calculated from the temperature dependences of the electron-transfer rates over the range 5-40°C are 1.7 ± 1.6 (His-48), 4.7 ± 0.9 (His-12), 5.4 ± 0.4 (His-116), and 5.6 ± 2.5 (His-81) kcal mol-1. Dynamic flexibility of the protein region containing His-48 may reduce the activation enthalpy with respect to the other more rigidly located derivatives.