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: 1987
• Subjects: Sperm whale
• Online Access: https://thesis.library.caltech.edu/11439/; https://thesis.library.caltech.edu/11439/2/Axup_AW_1987.pdf; 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) (a 5 Ru) derivatives of sperm whale myoglobin (Mb). The derivatives are a 5 Ru(His-48)Mb, a 5 Ru(His-12)Mb, a 5 Ru(His-116)Mb, and a 5 Ru(His-81)Mb. Pulsed laser excitation of the zinc myoglobin produces the long-lived and highly reducing triplet excited state ( 3 ZnP * ). Electron transfer from this triplet to the ruthenium, 3 znP * -Ru 3+ → ZnP + -Ru 2+ (ΔE° ≅ 0.8 V), was measured by time-resolved transient absorption techniques. The observed electron-transfer rates are 7.0 x 10 4 , 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 C α -C β 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 a 5 Ru(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 k et = 8 x 10 9 exp(-β(R-4)) s -1 , where β = 1.2 Å -1 and R - 4 ≥ 0 (R is the minimum e-e distance ...