Anne-Frances Miller believes enzymes are catalysts extraordinaire. Consider the following: The industrial process used to make the vast quantities of fertilizer necessary to support agriculture worldwide involves exposing nitrogen gas (N2) to temperatures in excess of 400 degrees Celsius at 200 atmospheres of pressure. This illustrates the difficulty of breaking the triple bond in dinitrogen (second in strength only to that of carbon monoxide). Meanwhile, in the roots of leguminous plants, bacterial enzymes are carrying out the same chemical conversion at room temperature under standard pressure.
|Anne-Frances Miller in front of one of her NMR machines.
This is the reason that Miller will never cease to be fascinated by enzymes. “Their ability to speed up chemical reactions by factors of millions, billions or more gives biology access to chemistry that would be useless at uncatalyzed rates,” she says.
However, Miller, an associate professor of chemistry at the University of Kentucky and director of the university’s nuclear magnetic resonance spectroscopy facility, is quick to point out that proteins should not get all of the glory. “Many of the most marvelous enzymes subcontract out the dirty work,” she says. “The most difficult chemistry is actually being executed by metal ions or organic cofactors. What the protein does is help select the proper substrate, focus the reactivity on the desired reaction and coordinate the reaction with other aspects of metabolism.”
That partnership between the protein and its cofactor forms the basis of Miller’s research interests. Using spectroscopic tools like NMR and electron paramagnetic resonance, which can reveal the details of the molecular interactions occurring at the interface of the protein and cofactor, Miller seeks to understand the mechanistic basis behind enzyme catalysis, particularly oxidation-reduction reactions.
And, by answering questions about, for example, how proteins guide the specificity of broadly reactive cofactors like metal ions or how a flavin’s chemical properties change when it becomes associated with a protein, Miller hopes to figure out one of the most enduring mysteries in enzymology: how proteins can both activate and control such powerful chemical reactions.
“Take dioxygen, for example,” Miller says. “Molecular oxygen is an extremely reactive molecule thermodynamically, but it also has a large kinetic barrier for activation. This is why it has accumulated to about 20 percent of our atmosphere. Because of that barrier, dioxygen holds a huge reservoir of potential energy.”
“Then, look at proteins,” she adds. “As reagents, they’re pretty mild-mannered— we even eat them for breakfast. How can proteins catalyze reactions with oxygen and not get burned up?”
Across the Border
|Overlay of heteronuclear single-quantum coherence spectra of uniformly 15N-labeled nitroreductase, collected at 37° C (red) and 4° C (black) showing a striking loss of dispersion attributable to conformational averaging.
Such a sense of wonder about the natural world has been a staple of Miller’s mindset since her youth in Toronto. She recalls that her scientific awakening occurred around the time she was 13 years old, when her family took her and a friend for a weekend naturalist program on Ontario’s Bruce Peninsula. During their hikes, Miller was fascinated by how much information the guides knew about every moss, plant and liverwort they passed, including tidbits such as the plant’s habitat range, what chemicals were inside it and how the indigenous people used it.
“I remember one foggy morning walk in particular,” she says. “We had heard a squawk in the distance above us, and one of our guides immediately told us that was a goshawk. That weekend revealed for me just how much information surrounds us, but we don’t notice, and so it passes us by. And I keep thinking how much richer our whole experience could be if we paid attention more.”
That weekend getaway eventually led to a vigorous pursuit of science projects, both for science fairs and personal curiosity; Miller even dabbled in some plant breeding, which led her to pursue a degree in molecular genetics at the University of Guelph in Ontario, Canada.
Along the way, Miller also began taking physics courses, because she found that most of the biology courses were too descriptive and she was eager to understand science at a deeper level; in fact, by the time she graduated in 1982, Miller was just one course short of a physics major.
At Guelph, Miller also got her first taste of NMR and EPR spectroscopy. “The notion that we could observe signals from single atoms or electrons was just amazing,” she says, “and it was a technology I wanted to learn more about.”
To do that, Miller crossed the border into the United States, following a career trajectory that included graduate studies at Yale University with Gary Brudvig, analyzing the assembly and mechanism of photosystem II, a postdoctoral position with William Orme-Johnson at the Massachusetts Institute of Technology and a second postdoctoral fellowship with Al Redfield at Brandeis University, conducting NMR studies on the conformation changes in the p21-Ras protein that contribute to tumor development. Her first independent appointment was at the Johns Hopkins University in 1992.
Her professional journey was far from a series of seamless transitions. For example, Miller considers her time in graduate school to have been quite rewarding but not entirely successful. “I had two projects that either didn’t prove interesting to anyone other than me, or, by the time they worked, someone else had published the result,” she admits, adding that she learned valuable lessons about what constitutes good science, and that helped her career immensely later on. “I am enormously grateful to Gary Brudvig for giving me independence so I could learn these important lessons before it was my career on the line. This was especially courageous of him considering that, at the time, his was.”
|2D PASS solid-state 15-NMR spectrum (bottom) and cross-polarization magic angle spinning 15N solid-state NMR spectrum of tetracetyl riboflavin labeled with 15N at positions N1, N3 and N5. For details, see Koder et al. 2006.
Other events were unforeseen, however, such as Miller having to leave her first postdoctoral position at MIT because her lab ran out of funding, forcing her to scramble to find a new lab to work in. This situation was made more difficult by the facts that her husband had just gotten a job in the Boston area and that Miller wasn’t a U.S. citizen and would have to leave the country if she didn’t find a U.S. Immigration and Naturalization Service-acceptable position very quickly.
And, while Miller did secure a position at Brandeis, two years later the “two-body problem” became an issue again. After many unsuccessful attempts at finding a suitable destination with her husband, Miller eventually received a job offer she simply could not refuse: assistant professor in the Johns Hopkins University chemistry department.
Although her long-distance “e-marriage” was trying, Miller had a fantastic time at Hopkins. “I had a chance to launch some very exciting studies and to work with fabulous colleagues; I would have loved to have been able to stay permanently.” Despite the best efforts by her colleagues, the university couldn’t find a way to open up a spot in the physics department for her husband. “After eight years, our family had reached a point where our first child was ready to start school, and we just had to be in the same city.”
That led Miller to a difficult professional decision— relocating her lab to the University of Kentucky in 1999 so her family could be together.
Heading into Orbit
While the nature of the projects in Miller’s group at the University of Kentucky varies to exploit the composition and interests of her lab members, she maintains an overall theme of combining principles of biophysics and spectroscopy to examine protein control over cofactor reactivity.
The lab focuses on two enzyme types: superoxide dismutases and enzymes that use flavins as cofactors. Superoxide dismutases, which metabolize toxic superoxide ions (O2-), regulate the reactivity of potentially reactive chemical species and are fairly well studied, providing a firm foundation for detailed studies of fundamental questions.
“That is not to say superoxide dismutase has no more new stories to tell, because it certainly has,” Miller says, noting some exciting work in which her lab provided the first mechanistic explanation as to why iron- or manganese-containing superoxide dismutases become inactive if their cofactor is exchanged with the opposing ion, even though the three-dimensional structures of the two enzyme types are basically superimposable.
Out of Focus: Language Barrier
While you won’t catch more than a hint of a Canadian accent in speaking with Miller these days, she admits to having had occasional communication “challenges” when she first moved from Guelph to New Haven, Conn. This led to one of her more bizarre graduate school experiences. One day, while returning from school, she was approached and accosted by a pair of youths who demanded her bicycle. “Their accent was so strong and foreign to me that I could barely understand them,” she says. Add in the fact that she came from a small, quiet college town, and she was not prepared for such a situation. “So rather than run away immediately (and lose my bike), I responded with a polite, if scared, refusal. Then they had trouble comprehending me. After several back-and-forth exchanges in which I can remember thinking I was completely crazy to be insisting on retaining my bicycle and repeating ‘I beg your pardon’ (because I still could not understand their English), instead of fleeing back up the street, one of them cracked a smile.” She says, “This whole conversation was probably the last thing they expected and in retrospect, it really was humorous. Once it had become a joke, they waved me on and I rode off. I would, nonetheless, not recommend this as a general strategy.”
Other recent spectroscopic analysis has revealed insights into how superoxide dismutase controls the movement of the electrons between the active site metal ion and substrate. “Proteins do not have good means of controlling electrons directly,” Miller says. “But we found that the big bridge by which superoxide dismutases regulate the sources and destinations of the transferred electrons is the protein’s exceptional control over protons, because the protons have a very big influence over where the electrons go.”
Miller chose enzymes that use flavins as cofactors as her second interest, because these cofactors, which resemble nucleotides, hearken back to the ancient RNA world and are likely the remnants of the evolutionary ancestors to enzymes. And, as organic molecules, not inorganic metal ions, they have different spectroscopic properties that enable Miller to ask a different set of questions.
Solid-state NMR, which, as implied by the name, examines samples that are solids or frozen solutions, can prevent the molecules under study from moving or reorienting. This allows orientation-dependent properties to be observed in the spectra, and, in Miller’s case, allows the three orientationally distinct components of the chemical shift to be resolved.
Miller has looked at the carbon and nitrogen atoms of the flavin ring system to complement solution NMR studies of the surrounding amino acids of the protein. Most importantly, the solid-state NMR results often can distinguish between effects on different orbitals of the flavin, resulting from different interactions between the flavin and the protein. With that information, she hopes to understand how different protein environments cause the bound flavin to emphasize different reactivities out of its inherently broad repertoire. Meanwhile, solution NMR studies of the surrounding protein address issues such as how some flavoenzymes like nitroreductase have such a broad substrate specificity range.
Beyond these studies, though, Miller is also busy trying to improve on the existing NMR and EPR technologies, so as to give them a broader and more cost-effective appeal.
In discussing her drive to do this, Miller reflects back on when she first came to the U.S. for graduate school. “At the time I left Guelph, there were very few positions available in Canada, as funding for universities was very tight,” she says. “My professors not only repaired laboratory equipment themselves, because they couldn’t afford to get it serviced, they built the equipment themselves as well.”
Considering the perilous nature of today’s economy, such memories resurface. “In a time of tightening budgets, there will be questions about the need to continue to run expensive NMR facilities,” she says, adding that the cost not only reflects the machines but the cryogens and reagents (like heavy isotopes of carbon and nitrogen) required to produce NMR-quality samples. While NMR holds many advantages as a tool for structure determination, it is weak when it comes to sensitivity because the magnetic moments of nuclei are quite small, thus, requiring large amounts of pure protein in each sample.
Some research groups have begun trying to alleviate the sensitivity problem by combining elements of NMR and EPR technology in a new application known as dynamic nuclear polarization. Rather than directly polarizing (or exciting) nuclear magnetic moments, DNP polarizes electrons first, as they have magnetic moments about 660 times that of the 1H magnetic moment. DNP then transfers that polarization to nearby nuclei. “So in theory,” says Miller, “you could have an NMR signal that’s 660 times more powerful than usual, which is mind-boggling.”
Thanks to a sabbatical she took, Miller, in collaboration with Thorsten Maly and Robert G. Griffin at MIT’s magnet lab, has tried to take DNP one step further. “Currently, DNP relies on added free radicals as bearers of the unpaired electrons,” she says, “but I realized that biology provides built-in radicals whose unpaired electrons can be used as sources of polarization. Many flavoproteins can be prepared with the flavin in a radical state, and the flavin molecule is bound in exactly the same way in each molecule. So we know where the polarization starts in every instance, in contrast with the random and uncontrolled locations of exogenous radicals.” Moreover, the flavin radical is often located in the enzyme’s active site.
“So, instead of having to analyze an entire protein, you can take a shortcut and focus your measurements just on the active site,” she continues. This “smart” DNP, as Miller refers to it, should make the technique more applicable than ever, as a researcher won’t need large quantities of protein or even a pure sample. Only protein molecules containing the flavin would be evident in a DNP-NMR spectrum.
Miller, A.-F. (2008) Redox Tuning Over Almost 1 V in a Structurally-conserved Active Site: Lessons from Superoxide Dismutase. Acc. Chem. Res. 41, 501-510.
Koder, R. L.,Walsh, J. D., Pometun, M. S., Dutton, P. L., Wittebort, R. J., and Miller, A.-F. (2006) 15N Solid-state NMR Provides a Sensitive Probe of Oxidized Flavin Reactive Sites. J. Am. Chem. Soc. 128, 15200–15208.
Vance, C. K. and Miller, A.-F. (1998) A Simple Proposal that can Explain the Inactivity of Metal-substituted Superoxide Dismutases. J. Am. Chem. Soc. 120, 461-467.
Miller, A.-F., Halkides, C. J., and Redfield, A. G. (1993) An NMR Comparison of the Changes Produced by Different Guanosine 5’-Triphosphate Analogs in Wild-type and Oncogenic Mutant p21ras. Biochemistry 32, 7367–7376.
Miller, A.-F. and Brudvig, G. W. (1989) Manganese and Calcium Requirements for Reconstitution of Oxygen-Evolution Activity in Manganese-Depleted Photosystem II Membranes. Biochemistry 28, 8181–8190.
Nick Zagorski (firstname.lastname@example.org) is a science writer at ASBMB.