Distance dependence of photoinduced long-range electron transfer in zinc/ruthenium-modified myoglobins

Abstract

An experimental investigation of the distance dependence of long-range electron transfer in zinc/ruthenium-modified myoglobins has been performed. The modified proteins were prepared by substitution of zinc mesoporphyrin IX diacid (ZnP) for the heme in each of four previously characterized pentaammineruthenium(II1) (a5Ru; a = NH,) derivatives of sperm whale myoglobin (Mb): a5Ru(His-48)Mb, aSRu(His-12)Mb, a5Ru(His-l 16)Mb, a5Ru(His-81)Mb. Electron transfer from the ZnP triplet excited state (,ZnP*) to Ru3+, 3ZnP*-Ru3t -+ ZnP+-Ru2+ (AEo -0.8 V) was measured by time-resolved transient absorption spectroscopy: rate constants (kf) are 7.0 X lo4 (His-48), 1.0 X IO2 (His-12), 8.9 X 10' (His-1 16), and 8.5 X IO' (His-81) s-' a t 25 OC. Activation enthalpies calculated from the temperature dependences of the electron-transfer rates over the range 5-40 O C are 1.7 i 1.6 (His-48), 4.7 i 0.9 (His-12), 5.4 i 0.4 (His-116), and 5.6 i 2.5 (His-81) kcal mol-'. Electron-transfer distances (d = closest ZnP edge to a5Ru(His) edge; angstroms) were calculated to fall in the following ranges: . The rate-distance equation is kf = 7.8 X lo8exp [-0.9l(d - it is generally assumed t h a t t h e rates of these transfers are determined to a large extent by the donor-acceptor separation and t h e nature of the intervening medi~m,l'-~' very few experiments have addressed these points in a systematic manner.'^^.''One approach t o t h e study of long-range electron transfer is to attach a redox-active complex t o a specific surface site of a structurally characterized heme or blue copper protein, thereby producing a two-site molecule with a fixed donor-acceptor distance. T h e redox-active complex t h a t has been employed successfully in several previous experiments is a5Ru2+/3+ (a = NH,), which covalently bonds to surface histidines.8-12 This complex can be attached by t h e reaction of a5Ru(OH,)2t with native protein under mild conditions, and t h e ruthenated protein can be purified by ion-exchange chromatography.In order t o probe distance effects on long-range electron transfer, we have replaced the heme in four ruthenated myoglobins (a5Ru(His-48)Mb; a5Ru(His-1 2)Mb; a5Ru(His-l 16)Mb; a5Ru-(His-81)Mb: Mb = sperm whale myoglobin)'O by zinc mesoporphyrin IX diacid (ZnP). The ZnP excited triplet state (3ZnP*) is a much more powerful electron donor t h a n a reduced heme (hE0(3ZnP*-Ru3+ -ZnP+-Ru2+) -0.8 V)," thereby allowing four different electron-transfer distances to be examined in a single protein molecule. Experimental SectionMaterials and Apparatus. Distilled water, filtered through a Barnstead Nanopure water purification system (No. 2794, specific resistance >18 Ma-cm), was used in the preparation of all aqueous solutions. 2-Butanone (MCB) was stored over aluminum oxide (Woelm neutral, Waters Associates) at 4 OC to prevent the accumulation of peroxides. All other reagents were used as received.Carboxymethylcellulose cation-exchange resin, CM-52 (Whatman, preswollen, microgranular), was equilibrated as indicated by the manu...

N‐(2‐Ferrocene‐ethyl)maleimide : a new electroactive sulphydryl‐specific reagent for cysteine‐containing peptides and proteins

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Photochemical Initiation of Electron Transfer Reactions

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Resonance effects in strongly exothermic long‐range electron transfer and their possible implications for the behaviour of site‐directed mutant proteins

“…Interference from double exchange ET from bridge group to photoexcited metalloporphyrin hole followed by ET from the excited donor electron to bridge cation radical, could here be envisaged. feasible due to the high negative EoS of the oxidized metalloporphyrins [1][2][3][4][5][6][7]. ET from Ru(lI) via the bridge group LUMO is not feasible due to poor redox potential match.…”

“…A number of the documented cases of long-range metaiioprotein ET have involved excited electronic states of the metal centres, corresponding to the sequence [1][2][3][4][5][6][7][8][9][10][11]25]:…”

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