Alkaline conformational transition and gated electron transfer with a Lys 79 → His variant of iso-1-cytochrome c

Swati Bandi, Saritha Baddam, Bruce E. Bowler

Research output: Contribution to journalArticlepeer-review

Abstract

To probe the mechanism of the alkaline conformational transition and its effect on the dynamics of gated electron transfer (ET) reactions, a Lys 79 → His (K79H) variant of iso-1-cytochrome c has been prepared. Guanidine hydrochloride denaturation monitored by circular dichroism and absorbance at 695 nm indicates that this variant unfolds from a partially unfolded state. The conformation of the wild type (WT) and K79H proteins was monitored at 695 nm from pH 2 to 11. These data indicate that acid unfolding is multi-state for both K79H and WT proteins and that the His 79-heme alkaline conformer is more stable than a previously reported His 73-heme alkaline conformer. Fast and slow phases are observed in the kinetics of the alkaline transition of the K79H variant. The pH dependence of the fast phase kinetic data shows that ionizable groups with pKa values near 6.8 and 9 modulate the formation of the His 79-heme alkaline conformer. The slow phase kinetic data are consistent with a single ionizable group with a pKa near 9.5 promoting the Lys 73-heme alkaline transition. In the broader context of data on the alkaline transition, ionization of the ligand replacing Met 80 appears to play a primary role in promoting the formation of the alkaline conformer, with other ionizable groups acting as secondary modulators. Intermolecular ET with hexaammineruthenium(II) chloride shows conformational gating due to both His 79-heme and Lys 73-heme alkaline conformers. Both the position and the nature of the alkaline state ligand modulate the dynamics of ET gating.

Original languageEnglish
Pages (from-to)10643-10654
Number of pages12
JournalBiochemistry
Volume46
Issue number37
DOIs
StatePublished - Sep 18 2007

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