Membrane Enzymes Involved in Energy Transduction;
Electron Transport Chains in E. coli and R. sphaeroides
Our laboratory studies the structure and function of cytochrome oxidase
and other membrane respiratory complexes with the goal to understand how
electron transfer is coupled to the generation of a transmembrane proton
electrochemical gradient. We are primarily interested in the structure
and function of membrane proteins that are proton pumps. Our efforts are
directed at several membrane enzymes that are components of bacterial
respiratory or photosynthetic electron transport systems. Of principle
interest are the members of the large respiratory oxidase superfamily
known as the heme-copper oxidases. This superfamily includes the mammalian
cytochrome c oxidase and many prokaryotic homologues. The structures of
two enzymes in this superfamily have been determined to atomic resolution
by X-ray diffraction techniques. The heme-copper oxidases caltalyze the
reduction of O2 and utilize the free energy liberated by this reaction
to pump protons electrogenically across the membrane bilayer (4 H+ / O2).
This generates transmembrane voltage and pH gradients, constituting the
protonmotive force. The protonmotive force is used to drive ATP synthesis,
active transport of solutes and other reactions. The structure of these
enyzmes show two putative proton-conducting channels and we are interested
in the roles of residues in these channels during the catalytic cycle.
The bacterial oxidases offer the opportunity to utilize the full array
of molecular genetics techniques in combination with spectroscopic methods
to address the catalytic mechanism of these enzymes. Single-turnover rapid
kinetics techniques are being utilized to examine these questions using
site-directed mutations in two members of the heme-copper oxidase superfamily.
These are the E. coli cytochrome bo3 ubiquinol oxidase and the Rhodobacter
sphaeroides aa3-type cytochrome c oxidase. In addition, we are utilizing
FTIR difference spectroscopy to identify specific amino acids directly
engaged in the catalytic mechanism.
One structural question that remains is the location of the binding
site for ubiquinol to cytochrome bo3. We are approaching this by using
a photoreactive analogue of quinol which covalently attaches to cytochrome
bo3 upon irradiation. Protein chemistry techniques, including mass spectroscopy,
will be used in this project. Another approach to address this question
is to use genetics methods by mapping mutants that confer resistance to
inhibitors that compete with ubiquinol at a common binding site.
In addition to the studies on the heme-copper oxidases, we are also
examining other respiratory enzymes that generate a protonmotive force.
These are the cytochrome bd ubiquinol oxidase from E. coli and the cytochrome
bc1 complex from Rhodobacter sphaeroides. We are in using techniques aimed
at obtaining structural information, including X-ray diffraction, as well
as examining aspects of the catalytic mechanisms of these enzymes.