Structure-function relationships in cytochrome f of oxygenic photosynthesis

Michael Vladimirovich Ponamarev, Purdue University

Abstract

Cytochrome f is the largest subunit of the cytochrome [special characters omitted] complex of oxygenic photosynthetic membranes. Cytochrome f serves as an electron acceptor from Rieske iron-sulfur protein, another subunit of the [special characters omitted] complex, and an electron donor to plastocyanin. The crystal structure of the lumen-side domain of cytochrome f revealed several unprecedented features and provided a basis for the structure-function studies (Martinez et al., 1994, 1996). The nature of one of the heme ligands, [special characters omitted]-amino group of the N-terminal Tyr residue, is unprecedented among all heme proteins. A prominent basic region on the cytochrome f surface was inferred to be a docking site for its redox partner plastocyanin. Another unique feature of cytochrome f is the highly ordered internal chain of the five water molecules, which has the properties of a proton conducting channel. The objective of the present studies was to test the functional significance of the cytochrome f structural features through site-directed mutagenesis and biophysical assay of cytochrome redox function both in vivo and in vitro. The effect of the neutralization of the cytochrome f basic surface region on its reaction with plastocyanin in vivo was very small. However, the Lys residues of the basic patch were critical for a high reaction rate between the two proteins in solution. A fast and efficient heterologous expression system for soluble holocytochrome f was developed in order to facilitate characterization of a large number of mutant variants. The E. coli expression system was used to measure redox and spectroscopic properties of the mutant proteins as well as for large scale purification of selected mutants for crystallographic studies. The function of the conserved H2O chain was tested in vivo in cytochrome f mutants in which the H-bonding environment of the internal water molecules was perturbed. The mutants showed a pronounced inhibition of rates of cytochrome f reduction and generation of the slow electrochromic bandshift in vivo, with no significant effect on the rate of cytochrome [special characters omitted] reduction. It was inferred from the observed non-concerted reduction of cytochromes f and [special characters omitted] that an equal rate of reduction of cytochromes f and [special characters omitted] is not required in the mechanism of quinol oxidation by the [special characters omitted] complex. Inhibition of the slow electrochromic phase implied a role of the cytochrome f water chain in electrogenic H + translocation through the complex. Disruption of the water chain in the non-phototrophic N168F mutant, revealed by the crystal structure, clearly demonstrated the functional significance of the water chain in cytochrome f. Mutagenesis studies of the cytochrome f N-terminal residues revealed a structural basis for the difference between the redox and spectroscopic properties of the cytochrome f from higher plants and cyanobacteria. The nature of the conserved N-terminal residues is one of the major factors that determine redox and spectroscopic properties of the cytochrome.

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Biophysics|Molecular biology|Biochemistry

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