Substrate binding and activation in the active site of cytochrome <Emphasis Type="Italic">c</Emphasis> nitrite reductase: a density functional study |
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Authors: | Dmytro Bykov Frank Neese |
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Institution: | (1) Institute of Physical and Theoretical Chemistry, University of Bonn, 53115 Bonn, Germany; |
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Abstract: | Cytochrome c nitrite reductase is a homodimeric enzyme, containing five covalently attached c-type hemes per subunit. Four of the heme irons are bishistidine-ligated, whereas the fifth, the active site of the protein,
has an unusual lysine coordination and calcium site nearby. A fascinating feature of this enzyme is that the full six-electron
reduction of the nitrite is achieved without release of any detectable reaction intermediate. Moreover, the enzyme is known
to work over a wide pH range. Both findings suggest a unique flexibility of the active site in the complicated six-electron,
seven-proton reduction process. In the present work, we employed density functional theory to study the energetics and kinetics
of the initial stages of nitrite reduction. The possible role of second-sphere active-site amino acids as proton donors was
investigated by taking different possible protonation states and geometrical conformations into account. It was found that
the most probable proton donor is His277, whose spatial orientation and fine-tuned acidity lead to energetically feasible, low-barrier protonation reactions. However,
substrate protonation may also be accomplished by Arg114. The calculated barriers for this pathway are only slightly higher than the experimentally determined value of 15.2 kcal/mol
for the rate-limiting step. Hence, having proton-donating side chains of different acidity within the active site may increase
the operational pH range of the enzyme. Interestingly, Tyr218, which was proposed to play an important role in the overall mechanism, appears not to take part in the reaction during the
initial stage. |
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