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Scientific Report 2006
Molecular Biology
Metalloenzyme Engineering
D.B. Goodin, C.D. Stout, S. Vetter, E.C. Glazer, R.F. Wilson, A. Annalora, A.-M. Hays
Our
goals are to understand the diverse reactivity of heme enzymes and to use that information
to generate engineered forms with novel catalytic properties. The primary hypothesis
that has driven these studies is that the chemical reactivity displayed by these
enzymes resides partially within the heme cofactor. One role of the protein is to
limit or direct the access of substrates to the active site in ways that result
in specific catalysis. In addition, many important examples exist in which the protein
directly modulates the activity of the heme. Thus, our goals are to delineate the
boundaries between these 2 roles for the protein and then use this information to
introduce sites where nonnative substrates interact with the heme cofactor in ways
that will induce new catalytic reactions. We use a number of techniques in structural
biology and spectroscopy and strategies of rational protein redesign and molecular
evolution.
One area of emphasis is the basic
physical, spectroscopic, and functional properties of heme enzymes. For example,
the FeIII/FeII and FeII/FeI redox couples of inducible nitric oxide synthase have
recently been measured by using direct cyclic voltammetry in organic films on graphite
electrodes. These studies allow easy measurement of electron transfer between the
enzyme and the electrode surface and have revealed the interconversion of several
coordination states of the heme. The results will complement ongoing studies in
which the enzymes are directly and homogeneously coupled to electrode surfaces by
using molecular wires.
In other research, we used cavity
complementation to introduce small-molecule binding sites near the active site of
a protein environment. This approach has provided new ways to test ideas about the
diversity of the functions of heme enzymes and is a useful tool for testing predictions
of protein-ligand interactions. For example, in a collaboration with B. Shoichet,
University of California, San Francisco, we completed a study in which compounds
in a database were docked into a buried engineered cavity that has an unusual specificity
for charged ligands. Using x-ray crystallography, we verified the accuracy of the
docking predictions for 15 of the top 16 compounds.
In other studies, we used synthetic
molecular wires, substrate analogs linked to photochemical or redox-active sensitizers,
to bind at the active site of cytochrome P450, peroxidases, and nitric oxide synthase.
These wires will be useful as reporters of the active-site environment and as triggers
to study reaction mechanisms. In collaboration with H.B. Gray, California Institute
of Technology, Pasadena, California, we recently solved structures of cytochrome
P450cam bound to 2 such wires. Marked changes in the protein structure
occurred near 2 helices that are similar to structural variations seen in mammalian
P450s, suggesting that the degree of structural plasticity in prokaryotic P450s
is similar to that of mammalian forms.
In other research, we removed the
proposed electron-transfer pathway from a peroxidase and replaced it with a solvent-filled
channel. We have designed surrogate molecular wires to replace the native pathway,
and we have shown that one of these binds the channel in a mode that is completely
analogous to the native structure. This technique will provide new ways to test
proposals about the specificity and structural requirements of this important structural
element. Finally, we are designing and synthesizing a class of cofactor-linked ruthenium-diamine
photosensitizers that are designed to specifically target nitric oxide synthase
at the pterin-binding site, allowing the role of the cofactor in catalysis to be
probed by direct-charge injection-withdrawal through the wire.
Publications
Brenk, R., Vetter, S., Boyce,
S.E., Goodin, D.B., Shoichet, B. Probing molecular
docking in a charged model binding site. J. Mol. Biol. 357:1449, 2006.
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