Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase.
Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins.
Marcia
Marco
M
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
Langer
Julian D
JD
Parcej
David
D
Vogel
Vitali
V
Peng
Guohong
G
Michel
Hartmut
H
D001426:Bacterial Proteins / Q000737:chemistry*; D003460:Crystallization; D003461:Crystallography; D011808:Quinone Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D011808:Quinone Reductases; C073412:sulfide quinone reductase