Mechanistic aspects of horseradish peroxidase elucidated through single-molecule studies

Many individual horseradish peroxidase (HRP) molecules were isolated and observed simultaneously by fluorescence microscopy in an array of 50 000 femtoliter chambers chemically etched into the surface of a glass optical fiber bundle. The substrate turnovers of hundreds of individual HRP molecules were readily analyzed, and the large number of molecules observed provided excellent statistics. In contrast to other enzymes used for single-molecule studies, the rates of product formation in the femtoliter array were, on average, 10 times lower than in bulk solution. We attribute this phenomenon to the particular redox-reaction mechanism of HRP that involves two separate steps of product formation. HRP first oxidizes fluorogenic substrate molecules like Amplex Red to radical intermediates. Two radical molecules subsequently undergo an enzyme-independent dismutation reaction, the rate of which is decreased when confined to a femtoliter chamber resulting in less product. This two-step reaction mechanism of the widely used Amplex Red, as well as other fluorogenic substrates, is often overlooked. The mechanism not only affects single-molecule studies with HRP but also bulk reactions at low substrate turnover rates.