Site-specific covalent attachment of heme proteins on self-assembled monolayers.

Naturally occurring hemin cofactor has been functionalized to introduce two terminal alkyne groups. This modified hemin has been successfully covalently attached to mixed self-assembled monolayers of alkanethiols and azide-terminated alkanethiols on gold electrodes using a Cu(I)-catalyzed 1,3-cycloaddition reaction. However these hemin-modified electrodes could not be used to reconstitute apomyoglobin on gold electrodes owing to the hydrophobicity of the alkane thiol self-assembled monolayer. Modification of existing techniques allowed covalent attachment of alkyne-terminated electroactive species onto mixed monolayers of azidothiols and carboxylatoalkanethiols on electrodes using the same Cu(I)-catalyzed 1,3-cycloaddition reaction. Apomyoglobin could be reconstituted using the hemin covalently attached to these hydrophilic electrodes. The electrochemical data, UV-vis absorption data, surface-enhanced resonance Raman spectroscopy data, and atomic force microscopy data indicate the presence of these modified myoglobin proteins on these electrodes. The direct attachment of the heme cofactor of these modified myoglobin proteins to the electrode allows fast electron transfer to the heme center from the electrode and affords efficient O(2)-reducing bioelectrodes under physiological conditions.