PNNL-13501 - Pacific Northwest National Laboratory

Study Control Number: PN99029/1357
Improving H2 Production by Rational Enzyme Redesign
Rick Ornstein
Because of the large potential for use of H2 as a clean fuel and alternative energy source, considerable recent effort has
been directed to improve the efficiency of hydrogenase mediated H2 production. Our focus is to develop the necessary
background capability to apply a rational enzyme redesign approach to increase the efficiency of this enzyme.
Hydrogenases can oxidize H2 to provide a microorganism with a source of strong reductants or generate hydrogen as
sinks of excess electrons. For example, anaerobic sulfate-reducing bacteria can grow on H2 as an electron donor with
sulfate and thiosulfate as terminal electron acceptors. Conversely, these same bacteria produce hydrogen upon
fermentative growth on pyruvate in the absence of sulfate.
Project Description
Although a growing number of three-dimensional x-ray
structures of hydrogenases have been solved by high
resolution, important ambiguities exist about the active
site structural details and much is unknown about the
fundamental mechanism of action(s). We employed stateof-the-art
computational and theoretical methods to begin
to resolve these ambiguities.
Introduction
Hydrogenase from Desulfovibrio gigas (D. gigas) has
been characterized by determining the crystal structure
and by a variety of spectral methods. The
crystallographic analysis of the enzyme has shown that
the active site is a bimetallic center that is attached to the
protein via four thiolates from cysteine residues. The
bimetallic center is known to be Ni-Fe. The Fe center is
also coordinated to three nonprotein ligands, two CN and
one CO (Scheme 1). The electronic and steric properties
of the bimetallic center and the attached nonprotein
ligands crucially influence enzymatic H2 heterolytic bond
cleavage, and remain uncertain. We employed state-ofthe-art
computation chemistry methods to address this
question.
Approach
The electronic and steric effects of the cysteines and
protein backbone were determined by density functional
theory calculations on a series of the model complexes:
-
(CO)(CN)2Fe(SR) Ni(SR) ,
2 2
(CO)(CN)(CNH)Fe(SR) Ni(SR) ,
2 2
-
X···(CO)(CN)2Fe(SR) Ni(SR) , and
2 2
X···(CO)(CN)(CNH)Fe(SR) Ni(SR) (R1, R2 = H, CH3; X
2 2
= H2O, NH3). Additional details are described in Nui and
Ornstein (submitted).
Results and Accomplishments
For the first model complex, the following SR
combinations were examined:
• all SCH3
• all SH
• Fe(SCH3)2Ni(SH)2
• Fe(SH)2Ni(SCH3)2.
The atomic spin density, charge, and total energy of each
structure were determined (see Table 1, Nui and
Ornstein). Our calculations indicate:
• The SH model for the cysteines in the actual [NiFe]
D. gigas hydrogenase is acceptable for an initial
investigation of the structures and reaction
mechanisms of the active sites,
(CO)(CN)2Fe(SR)2Ni(SR)2, although this simplified
model might underestimate the reactivity of the
active site of the [NiFe] hydrogenase.
• The protein backbone may contribute significantly to
the structural and electronic properties of the
bimetallic cluster of the active sites. However, these
steric distortions by the twist of the terminal
cysteines and the folding of the Ni-Fe cluster only
lead to a small destabilization of the Ni-Fe cluster,
and have no significant influence on the nonprotein
ligands, CO and CN.
Biosciences and Biotechnology 79