Jacobs, Bradley Anson (1991) Preparation, characterization, and intramolecular electron transfer in pentaammineruthenium-modified derivatives of cytochrome b5 and azurin. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06292007-133449
Wild-type, mutant, and deuteroporphyrin-substituted bovine cytochrome b5 have been modified with pentaammineruthenium (a5Ru) for intramolecular electron-transfer (ET) studies. The reactivity of the three surface histidines of the wild-type trypsinsolubilized protein (WT-b5) with [a5Ru(OH2)]2+ increases in the order His-15 < His-80 < His-26. Intramolecular ET rates from Fe(II) to Ru(IlI) have been measured by flash photolysis for a5Ru(His-26)-modified WT-b5, Ru(H26)WT-b5; mutant (asn-57 to Asp, Gln-13 to Glu, Glu-11 to Gln, His-15 to Asn, His-80 to Asn) lipase-solubilized cyt b5, Ru(H26)LM-b5; and deuteroporphyrin-substituted WT-b5, Ru(H26)DP-b5. The observed rates display a weak concentration dependence (0.5 - 3 µM protein, µ = 0.5 M sodium phosphate, ph 7.0 25°C): kET = 1.4(1)s[superscript-1], Ru(H26)WT-b5; 5.9(5) s[superscript-1], Ru(H26)LM-b5; and 0.2(1)s[superscript-1], Ru(H26)DP-b5. The rates do not directly correspond to differences in driving force (-0.08, -0.10, -0.13 eV) or edge-to-edge, donor-acceptor separation (12.1, 12, 12.9 A). Evaluation of the donor-acceptor electronic coupling (HAB) in terms of specific through-bond and through-space interactions in the intervening medium for Ru(H26)WT-b5 and Ru(H26)LM-b5 revealed a probable ET pathway consisting of eight covalent bonds from C[gamma] of His-26 to the end of the Leu-25 side chain, then a through-space jump (R = 3.8, 3.7 A) to the heme 2-vinyl. Ru(H26)DP-b5 lacks the 2-vinyl, requiring a longer jump (R = 4.5 A) to the heme 3-methyl. Because HAB is predicted to decay rapidly with R, the calculated rate constants (assuming [lambda] = 1.2 eV reflect the differences in R for the three derivatives: Ru(H26)WT-b5, 1.9 s[superscript-1]; Ru(H26)LM-b5, 4.4 s[superscript-1]; Ru(H26)DP-b5, 0.16 s[superscript-1]. The close agreement of calculated and observed rate constants indicates that the electronic coupling in this system is reasonably described by the (His-26)(Leu-25)(through-space jump to heme) pathway. The ET rate in a mutant (His-26 to Arg), trypsin-solubilized cyt b5 modified with a5Ru3+ at His-80 was also measured. As expected from the long donor-acceptor separation (21 A) and poor electronic coupling predicted by pathway analysis, kET was within experimental error of 0 s[superscript-1].
Azurin from Alcaligenes denitrificans was singly modified at His-83 with asRu3+; the other surface histidine (32) was not successfully modified. The intramolecular ET rate from Ru(II) to Cu(II) for Ru(H83)Az was measured (1.0(3) s[superscript-1]) and was found to be comparable to that reported for the analogous ruthenium-modified P. a. azurin. These rates are slower than would be expected for a similar distance in cytochrome c. The application of a pathway theoretical model to both the P. a. and A. d. azurin systems showed no evidence that the ß-sheet secondary structure of azurin precludes effective electronic coupling. This suggests that weak electronic coupling to the copper site itself is responsible for the low kET's in azurin.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||10 September 1990|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||23 Jul 2007|
|Last Modified:||26 Dec 2012 02:54|
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