| Abstract
| - The kinetics of electron transfer from reduced high-potential iron−sulfur protein (HiPIP) tothe photooxidized tetraheme cytochrome c subunit (THC) bound to the photosynthetic reaction center(RC) from the purple sulfur bacterium Allochromatium vinosum were studied under controlled redoxconditions by flash absorption spectroscopy. At ambient redox potential Eh = +200 mV, where only thehigh-potential (HP) hemes of the THC are reduced, the electron transfer from HiPIP to photooxidized HPheme(s) follows second-order kinetics with rate constant k = (4.2 ± 0.2) 105 M-1 s-1 at low ionic strength.Upon increasing the ionic strength, k increases by a maximum factor of ca. 2 at 640 mM KCl. The roleof Phe48, which lies on the external surface of HiPIP close to the [Fe4S4] cluster and presumably on theelectron transfer pathway to cytochrome heme(s), was investigated by site-directed mutagenesis. Substitutionof Phe48 with arginine, aspartate, and histidine completely prevents electron donation. Conversely, electrontransfer is still observed upon substitution of Phe48 with tyrosine and tryptophan, although the rate isdecreased by more than 1 order of magnitude. These results suggest that Phe48 is located on a key proteinsurface patch essential for efficient electron transfer, and that the presence of an aromatic hydrophobicresidue on the putative electron-transfer pathway plays a critical role. This conclusion was supported byprotein docking calculations, resulting in a structural model for the HiPIP-THC complex, which involvesa docking site close to the LP heme farthest from the bacteriochlorophyll special pair.
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