Fluorescence is also the technique that has been used in the highest new entry in the current Hot Ten, at #3, whose lead author is Sunney Xie of the Department of Chemistry and Chemical Biology at Harvard University. This noteworthy paper reports a new way of probing the relationships between structure and dynamics within an individual enzyme by means of electron transfer studies. The method is complementary to fluorescence energy transfer.

Paper #3 reports the fluctuations in distance between molecular centers within what is in effect a complex protein molecule, and it takes us one step further along the road to a full understanding of the molecular dynamics of the living cell. And how has Xie’s group achieved this remarkable step forward? The answer is by using fluorescence to examine electron transfer between flavin and tyrosine centers in the enzyme flavin reductase.

Electron transfer is highly dependent on the distance between the donor and acceptor moieties, and it has been observed by measuring fluorescence lifetimes, but not on a single-protein molecule. Xie has shown that the technique can be extended to highly complex molecules. The flavin enzyme chosen for study in #3 has an isoalloxiazine surrounded by three tyrosines, which are potential electron donors, located at various distances. By making mutant enzymes in which these tyrosines were replaced by serine groups, it was possible to identify the one that acted as the electron donor. (As might be expected, it was the one nearest to the isoalloxazine group.) To carry out the necessary observations the enzyme was tethered to a quartz surface by a biotin-streptavidin link.

An enzyme functions as a catalyst and its modus operandi is to seize a target molecule, modify it chemically, and then release it, all of which require the proteins from which the enzyme is made to undergo various conformational changes. Electron transfer is clearly an important part of such a process and Xie has shown that even this transient process is now observable by his sophisticated techniques.

Xie has published two follow-up papers to #3: the first was a theoretical one which proposed a model to explain the fluctuation of the distance between the electron transfer donor and acceptor pair within a protein (see S.C. Kou and X.S. Xie, Physical Review Letters, 93[18]:180603, 2004); the second, and more recent one, reports real-time experimental observations on a different system (fluorescein-to-tyrosine) and reveals the fluctuation of the donor-acceptor distance in this single-protein complex (see W. Min, et al., Physical Review Letters, 94[19]:198302, 2005). On the basis of this observation Xie remarks that it supports his belief that he is observing a general phenomenon.

More recently Xie’s group has been working on the enzymatic turnover rate of a single-enzyme molecule fluctuating over a broad range of time scales—a paper on the work has been submitted to Science. "We have proved that the conventional Michaelis-Menten equation also holds for the ever-fluctuating enzyme under most conditions," he tells Science Watch. The Michaelis-Menten equation generally provides a good description of the experimentally observed reaction rates of enzyme reactions, and this reconciles single-molecule and ensemble experiments.