Mutational and biophysical analysis suggests that an intracellular COOH-terminal domain of the large conductance Ca 2؉ -activated K ؉ channel (BK channel) contains Ca 2؉ -binding site(s) that are allosterically coupled to channel opening. However the structural basis of Ca 2؉ binding to BK channels is unknown. To pursue this question, we overexpressed the COOH-terminal 280 residues of the Drosophila slowpoke BK channel (Dslo-C280) as a FLAG-and His 6-tagged protein in Escherichia coli. We purified Dslo-C280 in soluble form and used a 45 Ca 2؉ -overlay protein blot assay to detect Ca 2؉ binding. Dslo-C280 exhibits specific binding of 45 Ca 2؉ in comparison with various control proteins and known EF-hand Ca 2؉ -binding proteins. A mutation (D5N5) of Dslo-C280, in which five consecutive Asp residues of the ''Ca-bowl'' motif are changed to Asn, reduces 45 Ca 2؉ -binding activity by 56%. By electrophysiological assay, the corresponding D5N5 mutant of the Drosophila BK channel expressed in HEK293 cells exhibits lower Ca 2؉ sensitivity for activation and a shift of Ϸ؉80 mV in the midpoint voltage for activation. This effect is associated with a decrease in the Hill coefficient (N) for activation by Ca 2؉ and a reduction in apparent Ca 2؉ affinity, suggesting the loss of one Ca 2؉ -binding site per monomer. These results demonstrate a functional correlation between Ca 2؉ binding to a specific region of the BK protein and Ca 2؉ -dependent activation, thus providing a biochemical approach to study this process.

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The roles of aromatic core residues in regulating the reduction potential, the enthalpy and entropy of reduction, and the self-exchange rate constants for electron-transfer reactions for the prosthetic [Fe4S4]3+/2+ cluster of Chromatium vinosum high potential iron protein (HiPIP) have been addressed by a combination of site-directed mutagenesis, high field NMR (EXSY) experiments, and variable temperature spectrochemical redox titration measurements. Minimal changes are observed following nonconservative mutation of residues Tyr19, Phe48, and Phe66. Apparently these hydrophobic residues play only a minor role in defining the electronic properties of the cluster. These data support a model, first defined from results obtained on Tyr19 mutant HiPIP's [Agarwal, A., Li, D., & Cowan, J.A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9440-9444], in which the aromatic core restricts solvent accessibility and thereby stabilizes the oxidized [Fe4S4]3+ cluster.

No abstract

A number of point mutations of the conserved aromatic residue phenylalanine 66 (Phe66Tyr, -Asn, -Cys, -Ser) in Chromatium vinosum high-potential iron sulfur protein have been examined with the aim of understanding the functional role of this residue. Nonconservative replacements with polar residues have a minimal effect on the midpoint potential of the [Fe4S4]3+/2+ cluster, typically < +25 mV, with a maximum change of +40 mV for Phe66Asn. With the exception of the Phe66Tyr mutant, the oxidized state was found to be unstable relative to the recombinant native, with regeneration of the reduced state. The pathway for this transformation involves degradation of the cluster in a fraction of the sample, which provides the reducing equivalents required to bring about reduction of the remainder of the sample. This degradative reaction proceeds through a transient [Fe3S4]+ intermediate that is characterized by typical g values and power saturation behavior and is prompted by the increased solvent accessibility of the cluster core in the nonconservative Phe66 mutants as evidenced by 1H-15N HMQC NMR experiments. These results are consistent with a model where the critical role of the aromatic residues in the high-potential iron proteins is to protect the cluster from hydrolytic degradation in the oxidized state.