Structural analysis of alternative complex III in the photosynthetic electron transfer chain of Chloroflexus aurantiacus.

The green photosynthetic bacterium Chloroflexus aurantiacus, which belongs to the phylum of filamentous anoxygenic phototrophs, does not contain a cytochrome bc or bf type complex which is found in all other known groups of phototrophs. This suggests that a functional replacement exists to link the reaction center photochemistry to cyclic electron transfer as well as respiration. Earlier work identified a potential substitute of the cytochrome bc complex, now named alternative complex III (ACIII), which has been purified from C. aurantiacus, identified, and characterized. ACIII functions as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain, and a related but distinct complex functions in respiratory electron flow to a terminal oxidase. In this work, we focus on elucidating the structure of photosynthetic ACIII. We found that ACIII is an integral membrane protein complex of approximately 300 kDa that consists of eight subunits of seven different types. Among them, there are four metalloprotein subunits, including a 113 kDa iron-sulfur cluster-containing polypeptide, a 25 kDa penta-heme c-containing subunit, and two 20 kDa monoheme c-containing subunits in the form of a homodimer. A variety of analytical techniques were employed in determining the ACIII substructure, including HPLC combined with ESI-MS, metal analysis, potentiometric titration, and intensity analysis of heme staining SDS-PAGE. A preliminary structural model of ACIII is proposed on the basis of the analytical data and chemical cross-linking in tandem with mass analysis using MALDI-TOF, as well as transmembrane and transit peptide analysis.

Purification and characterization of cytochrome c(6) from Acaryochloris marina.

Cytochrome c(6), (cyt c(6)) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E(m)) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.