Three-Dimensional Structures of Biological 
Macromolecules 
Johann Deisenhofer, Ph.D. — Investigator 
Dr. Deisenhofer is also Regental Professor and Professor of Biochemistry, and he holds the Virginia and 
Edward Linthicum Distinguished Chair in Biomolecular Science at the University of Texas Southwestern 
Medical Center at Dallas. He was born and educated in Germany. His Ph.D. research in protein 
crystallography was done at the Max Planck Institute for Biochemistry, Martinsried, and at the Technical 
University of Munich. As a postdoctoral fellow and as a staff scientist at the Max Planck Institute, he 
continued his structural analysis of biological macromolecules by x-ray crystallography. He has received 
several honors for his structure analysis of a photosynthetic reaction center, including the 1988 Nobel 
Prize in chemistry, which he shared with Hartmut Michel and Robert Huber. 
MY colleagues and I study the three- 
dimensional structures of proteins to un- 
derstand their folding, structural stability, and 
function. We are particularly interested in 
protein-pigment complexes catalyzing photo- 
chemical energy conversion, energy transfer, and 
electron transfer, and in membrane-spanning and 
membrane-associated proteins. 
Cytochrome b/ Cj complex 
The b/Cj complexes (also called ubiquinol- 
cytochrome c oxidoreductases) are integral 
membrane proteins that play crucial roles in pho- 
tosynthesis and cell respiration. Their function in 
these fundamental processes is to oxidize quinols 
to quinones and to transfer electrons and protons 
through the membrane. The electrons go to cy- 
tochrome c, and the protons build up an electro- 
chemical gradient across the membrane, which, 
for example, drives the synthesis of ATP. 
Photosynthetic purple bacteria have the sim- 
plest b/c, complexes, consisting of only three or 
four different protein subunits with three heme 
groups and an iron-sulfur cluster as cofactors. 
Photosynthetic reaction centers and b/c, com- 
plexes cooperate in the bacterial inner mem- 
brane; b/Ci complexes occur at a concentration 
significantly lower than that of reaction centers 
and are therefore more difficult to isolate. David 
Knafif's research group (Texas Technical Univer- 
sity, Lubbock) succeeded in purifying the b/Cj 
complex from Rhodospirillum ruhrum. In col- 
laboration with this group we have prepared this 
complex in milligram quantities with high pu- 
rity. This preparation serves as the starting mate- 
rial for crystallization experiments. 
Mitochondria of higher organisms have in their 
inner membranes b/Cj complexes consisting of at 
least 10 different polypeptide chains, 3 of which 
are similar to those in the b/Cj complex of purple 
bacteria. We collaborate with Chang-An Yu and 
his colleagues (Oklahoma State University, Still- 
water), who produced large crystals of the b/Cj 
complex from beef heart mitochondria. Prelimi- 
nary diffraction experiments at the Cornell High 
Energy Synchrotron Source (CHESS) were encour- 
aging, and we hope to begin a structure analysis at 
medium resolution (approximately 5 A) in the 
near future. 
DNA Photolyase 
Light energy plays an important role in reac- 
tions other than photosynthesis. The molecular 
machinery that enables cells to repair DNA dam- 
aged by ultraviolet (UV) light includes an en- 
zyme that uses light energy to drive the repair of 
one frequent type of damage caused by UV irra- 
diation: the crosslinking of two neighboring thy- 
mine bases in a strand of DNA. Most of these 
crosslinks are in the form of cyclobutane rings 
connecting four carbon atoms, two from each 
thymine base. The enzyme DNA photolyase can 
locate and bind to such defects, and upon input 
of light of suitable wavelength (300-500 nm) 
cleave the carbon-carbon bonds between the 
bases, thus repairing the damage. 
DNA photolyase has been found in prokaryotes, 
eukaryotes, and archaebacteria. Aziz Sancar and 
his colleagues (University of North Carolina at 
Chapel Hill) sequenced, overexpressed, and puri- 
fied the enzyme from Escherichia coll. It consists 
of a single polypeptide chain of 471 amino acids 
and two cofactors — a flavin-adenine dinucleo- 
tide (FAD) and 5, 10-methenyltetrahydrofolate. 
The FAD cofactor fully reduced to FADH2 is es- 
sential for the enzyme's function; the folate acts 
as a light-harvesting antenna. 
Despite significant problems with the en- 
zyme's tendency to denature, we were able to 
crystallize DNA photolyase from E. coli in two 
crystal forms; both forms difi'ract x-rays to at least 
2.8-A resolution. We collected x-ray diffraction 
intensity data from one of these crystal forms; to 
solve the phase problem, experiments are under 
way to bind heavy-atom compounds to the pro- 
tein in the crystal. We are also trying to crystallize 
the enzyme in complex with a substrate, a five- 
nucleotide piece of single-stranded DNA contain- 
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