Investigation of electron transfer in the [alpha]-helical protein cytochrome b [subscript 562]

Citation

Farrow, Neil (1999) Investigation of electron transfer in the [alpha]-helical protein cytochrome b [subscript 562]. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/8mrb-7d44. https://resolver.caltech.edu/CaltechETD:etd-02082007-151616

Abstract

Electron transfer rates in the [alpha]-helical protein cytochrome b[562] are investigated using the photoexciteable luminescent labelling compound ruthenium(II)bis(2,2'-bipyridine)(imidazole) attached to surface accessible histidine residues. Three residues on helices A and D and two on helix C were replaced with histidine by site directed mutagenesis to form eight mutant proteins which, together with a wild-type histidine on helix C, were labelled with ruthenium(II)bis(2,2'-bipyridine)(imidazole). Laser excitation of ruthenium(II)bis(2,2'-bipyridine)(imidazole) formed the highly reducing species *ruthenium(II)bis(2,2'-bipyridine)(imidazole) which was either quenched by the heme directly and the back rate (k[b][ET]) to the RuIII monitored by transient absorbance, or was oxidized by an external quencher and the rate from the reduced protein then monitored. Mutants spanned a metal-metal distance range from 14.2 Å to 25 Å with the rates ranging from 2.5 x 10[2] s[-1] to 2.2 x 107 s[-1]. A ß value of 1.09 Å[-1] is calculated from the data which agrees well with data from azurin and cytochrome c. The results show that the [alpha]-helices together with the extended heme plane provides an efficient homogenous barrier to electron transfer. A mutant of cytochrome b[562] with a covalently attached heme via one thioether linkage was expressed. The unfolding characteristics of this protein show it to be more stable to guanidinium unfolding than wild-type protein. The electron transfer rate for this mutant was identical to wild-type indicating minimal perturbation of the heme binding pocket by the mutation. It can be inferred that the side chain of the carboxyl terminal arginine residue and not Arg98 is responsible for stabilizing the His102 ligand histidinate charge.

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