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Priestley Medal address 2020: The road less traveled—for love of detection, discovery, and all things radical in nature

by Joanne Stubbe
March 23, 2020 | A version of this story appeared in Volume 98, Issue 11


This is a photo of JoAnne Stubbe.
Credit: Gretchen Ertl
Credit: Courtesy of JoAnne Stubbe
Because the ACS spring national meeting in Philadelphia was canceled, JoAnne Stubbe was not able to present her Priestley Medal Address. These are the slides she would have presented and will present when the talk is rescheduled at the ACS fall meeting.

Let me start by saying that I am grateful for having been paid to pursue my passion for so many years and more than a little taken aback by being named the Priestley Medal winner this year. While I have delivered hundreds of lectures, giving an after-dinner talk about myself that is entertaining and interesting, for me, a scientist’s scientist, is impossible. My task tonight is to describe a little about myself, how I became a biochemist interested in catalysis, and my lab’s scientific contributions. [Editor’s note: Because the ACS spring national meeting in Philadelphia was canceled due to the coronavirus outbreak, Stubbe did not deliver this talk during the awards dinner as planned. ACS is rescheduling her address for the fall meeting.] My career exemplifies the importance of collaboration. Without intellectual and experimental contributions from many students, postdocs, and collaborators, our understanding of all things radical in nature would be diminished.


Priestley Medal address 2020: The road less traveled—for love of detection, discovery, and all things radical in nature

I did not get a chemistry set when I was young, and I hated explosions! As a child, I spent my summers in a tar-paper shack on a lake in southern New Hampshire. We had an outhouse, no running water, and an icebox—yes, blocks of ice kept our food cold. I used to take a butterfly net to bed at night to catch the bats that swooped around me. My fondest memories are of loons crying at dawn.

I loved catching crawfish in the lake and capturing hellgrammites with my grandfather in trout streams. I sold them to fishermen for bait, my first and only encounter with entrepreneurship. I loved fishing. I still remember the day my brother, John, caught the largest smallmouth bass ever taken from Deering Reservoir. My job was to get it into the boat and then to sit on it so it wouldn’t jump out. I loved and still love the woods, wildflowers, birds, sounds, and smells.

How did I get from there to here? I come from a family of teachers. My dad was a mathematics professor and computer scientist, and my mom was a homemaker who later taught high school. My family encouraged me to find and pursue my interests and to be satisfied with my best efforts. In retrospect, both my mother and grandmother were important role models based on their resilience and their seemingly boundless energy. I must have inherited these genes. My dad introduced me to tournament bridge (at age 9), chess, punch cards and the IBM 1620 computer, tennis, and basketball. Basketball was a particular favorite: I used to sneak the radio to bed at night to hear Johnny Most broadcast the Celtics games!

Photo of JoAnne Stubbe (far right) and her three siblings.
Credit: Courtesy of JoAnne Stubbe
JoAnne Stubbe (far right) and her siblings enjoyed fishing in the summer.

I went to Classical High School, a public school in Worcester, Massachusetts. Important influences were my high school chemistry teacher, Mr. McQue, who provided me with a basis for understanding color and crystals, and two poets: Bob Dylan and Robert Frost. I remember hearing Robert Frost read his poetry at College of the Holy Cross while I was in high school. Both poetry and art have been important influences on my choices in life. The painting Christina’s World by Andrew Wyeth shows how I felt for most of my career.

I am grateful to two faculty mentors who taught me the scientific method and instilled a love of chemical mechanisms in me. Ed Trachtenberg, a chemist at Clark University who trained with P. D. Bartlett, taught me organic chemistry. He let me work in his lab for three summers supported by the National Science Foundation’s Research Experiences for Undergraduates program. How lucky I was to be paid to continually learn new stuff. I also carried out undergraduate research at the University of Pennsylvania with Ed Thornton, a chemist trained with Gardner Swain. The central role of mechanisms in my independent research career was set by these experiences. I also realized that I enjoyed messing around in the lab and loved the detective work. Without Dr. Thornton’s push to apply to graduate school at the University of California, Berkeley, I would not be giving this lecture today. Where you go to school does make a difference.

This is a photo of the painting Christina's World by Andrew Wyeth.
Credit: © 2020 Andrew Wyeth/Artists Rights Society (ARS), New York
The Museum of Modern Art describes Christina’s World, painted by Andrew Wyeth, as "more a psychological landscape than a portrait, a portrayal of a state of mind rather than a place."

In graduate school at Berkeley from 1968 to 1971, I was introduced to radicals and mechanism-based inhibitors. Berkeley was a life-changing experience. It was a time of upheaval: the draft, the Vietnam War, tear gas dumped on campus by helicopters in 1969, Latimer Hall’s ventilation system filled with butyric acid. Ronald Reagan was governor of California. He considered student protesters to be radicals: highly reactive, out of control, bent on destruction. However, as a participant in the protests, I realized how little provocation it took on either side to evoke uncontrolled behavior.

In biology today, radicals (reactive oxygen and nitrogen species, ROSs and RNSs) evoke the same response: in this case, the radicals are highly reactive molecules leading to destruction. They damage DNA, which, if left unrepaired, mutates and contributes to the aging process and disease. However, as with student protesters, everything in biology is also about homeostasis (regulation).

Graduate school also introduced me to rational drug design. All organic chemistry students were required to give a seminar on a topic of their choice to advance to candidacy for a PhD. Most students chose a topic related to synthesis or mechanism. I chose an unconventional topic based on a 1965 paper by D. J. Tipper and J. L. Strominger (Proc. Natl. Acad. Sci. U.S.A., DOI: 10.1073/pnas.54.4.1133). The thesis of the paper was that the antibiotic penicillin looked like acyl-d-alanine-d-alanine, which is involved in a transpeptidation reaction essential for the formation of a crosslinked-peptidoglycan bag that surrounds bacteria and is necessary for their survival. Penicillin inhibited this process and was one of the first examples of a mechanism-based inhibitor!

In graduate school, I worked with George Kenyon, one of the first hires in chemistry at Berkeley to work at the interface with biology. I worked on the enzyme pyruvate kinase, which you could buy from Sigma. I learned self-sufficiency and had fun figuring out the synthesis to make enol phosphates, which I used to study the specificity and stereochemistry of several enzymes. I took a course taught by Jack Kirsch and Daniel Koshland in the biochemistry department, in which mechanisms were applied to understanding enzymatic transformations. I became excited about the new frontier in chemistry: catalysis by enzymes. More important, I learned a lot about life as well as chemistry.

I still, however, lacked an important component for survival in the real world and the chemistry world: self-confidence. I was briefly a postdoctoral fellow with Julius Rebek at the University of California, Los Angeles, where I worked on the total synthesis of LSD, starting from tryptophan. There, I met Bill Moomaw, a faculty member at Williams College, who was on sabbatical. Williams is an outstanding undergraduate institution and was a bastion of maleness that had just opened its doors to women undergraduates in 1970. Bill asked me to apply for a job at Williams. During my job interview, I gave a seminar, not on my research but on the mechanism of penicillin. It was enthusiastically received, and I was offered a job.

Photo of Christopher Walsh and JoAnne Stubbe laughing at a celebration for the Welch Award.
Credit: Brian Phillips
JoAnne Stubbe and Christopher Walsh laugh together at a celebration for their shared Welch Award in 2010.

For a good chunk of the 1970s, I scrambled to learn physical chemistry and biochemistry and learn about teaching from the masters. I received an R01 grant from the National Institutes of Health to work on mechanism-based inhibitors, the first ever in the Chemistry Department at Williams!

The chemistry building had five floors. My office was on the fifth floor, and the only women’s bathroom was in the basement. I learned resourcefulness and carried around a “WO” sign that I placed in front of the “MEN” sign on the bathroom door so that I could use any one I pleased.

I enjoyed working with undergraduates: it was so easy to excite them about science! My experiences at Williams increased my teaching skills and self-confidence. With encouragement from Chris Walsh (then a young faculty member at the Massachusetts Institute of Technology), I applied for a leave of absence from Williams to work with Robert Abeles in the Biochemistry Department at Brandeis University. I wanted to learn how to purify proteins so that I could work on my own ideas. Brandeis had an electrifying environment with Abeles and William Jencks and a focus on understanding enzyme mechanisms and their basis for catalysis. I was in heaven. I really finally found my passion!

I was attracted to Abeles’s lab because of his interest in the development of mechanism-based inhibitors and his focus on the enzyme diol dehydratase, an adenosylcobalamin-containing enzyme that converted propanediol (which has a cis diol like the ribose of a nucleotide) to propanal and was proposed to involve radical chemistry. While I was in Abeles’s lab, B.-M. Sjöberg, P. Reichard, A. Gräslund, and A. Ehrenberg reported the observation and characterization of the first protein radical in an enzymatic system, a tyrosyl radical in the Escherichia coli ribonucleotide reductase (RNR) (J. Biol. Chem. 1978, 253, 6863).

Abeles’s interest in the adenosylcobalamin-dependent diol dehydratase, William Beck’s discovery of an adenosylcobalamin-dependent RNR, and the tyrosyl radical in the E. coli RNR all suggested to me the importance of unprecedented radical-based chemistry in RNRs, enzymes known to play an essential role in making the building blocks of DNA in all organisms. L. Thelander, F. Eckstein, and coworkers had also reported that 2′-substituted nucleotide analogs were inhibitors of RNR, although their mechanism—or mechanisms—was unknown (J. Biol. Chem. 1976, 251, 1398). Together, these studies suggested that RNR would provide an exciting opportunity to discover unprecedented radical chemistry and that it would potentially be an excellent therapeutic target.

Structure of the DNA-cleaving drug bleomycin.
JoAnne Stubbe collaborated with John Kozarich to study the mechanism of the DNA-cleaving drug bleomycin.

I decided not to return to Williams and obtained a job as an assistant professor in the Department of Pharmacology at Yale School of Medicine. I was hired at the same time as John Kozarich. We shared a tiny office and a lab with almost no ventilation. To avoid killing each other, we collaborated to study the mechanism of the clinically used antitumor antibiotic bleomycin. We established that this natural product mediated DNA strand cleavage by C1′ and C4′ radical chemistry in the presence of iron(II) and oxygen and that one bleomycin molecule was sufficient for double-strand DNA cleavage.

I also discovered that RNR catalyzes hydrogen atom abstraction from the 3′-carbon of the ribose ring of its nucleotide substrate. John taught me the nucleotide chemistry required to make these isotopically labeled molecules, which allowed our discoveries. We had fun! My self-confidence increased, and I applied for and obtained a job in the Department of Biochemistry at the University of Wisconsin–Madison. It was there that I attracted my first graduate students, and after 11 years as an assistant professor, I received tenure.


There were many things I loved about Madison: orange custard chocolate-chip ice cream from Babcock Hall; teaching with and learning from Mo Cleland about steady-state kinetics and isotope-effect measurements in enzymatic systems; Helmut Beinert, the father of FeS cluster biochemistry; Perry Frey and his inspirational studies on lysine aminomutase, including its importance in the radical SAM superfamily story involving more than 150,000 reactions in biology, all involving radicals; the Raetz family (Madeline, Lizzy, Jackie, and Chris); and even the pie-in the-face bet!

I was recruited to MIT in 1987. I am a New Englander at heart, and my family lives in Massachusetts. Remember Deering Reservoir and the big bass? While both the RNR and bleomycin studies were well underway, my move to MIT in 1987 was beyond transformative. The intensity and intellectual energy of this institution allowed my group to experience science in ways I never could have imagined. New directions and discoveries never stopped. While MIT is a tough place, I still pinch myself each day when I look at the MIT website and realize how lucky I have been to be here.

Structure of gemcitabine.
The cancer drug gemcitabine is a mechanism-based inihibitor of ribonucleotide reductase.

Finally for some science (indulge me): RNRs are a paradigm for radical-mediated transformations and a target of new therapeutics. RNRs in all organisms convert ribonucleotides (RNA building blocks) to deoxyribonucleotides (DNA building blocks) de novo. A single enzyme reduces four substrates (cytidine, uridine, adenosine, and guanosine 5′-diphosphates, collectively known as NDPs) to the deoxyribonucleotide products, dNDPs. Because the ratios of the building blocks and their absolute amounts are central to the fidelity of DNA replication and repair, RNRs are exquisitely regulated. Given the central role of human RNRs in nucleic acid metabolism, it is not surprising that they are targeted by five clinically used cancer therapeutics, including gemcitabine, a mechanism-based irreversible inhibitor. Ultimately, RNR inhibition contributes to cell cytotoxicity via DNA damage and replication stress and their downstream consequences.

There are three classes (I, II, III) of RNRs and five subclasses of the class I RNRs (Ia–Ie). Classification is based on the unique metallocofactors they possess. I will focus on the class Ia RNRs, the most extensively studied. The active form involves two subunits, α and β, that form a dynamic and asymmetric α2β2 complex. The active site of RNRs resides in a conserved architecture, the α subunit, which houses three essential cysteines. The reducing equivalents required to form dNDP products are provided by oxidation of two cysteines to a disulfide, which must then be rereduced before additional turnovers. The third cysteine is transiently oxidized to a thiyl radical, essential for initiation of NDP reduction by removal of the hydrogen atom from the C3′ position of the ribose ring. The chemically and kinetically competent thiyl radical was discovered by our rapid kinetic studies on the adenosylcobalamin-dependent class II RNR (Science 1996, DOI: 10.1126/science.271.5248.477). This observation and the structural homology in the active site of all three RNR classes strongly support the importance of protein thiyl radicals in catalysis.

Photo of JoAnne Stubbe (seated) with her first three graduate students at the University of Wisconsin.
Credit: Courtesy of JoAnne Stubbe
JoAnne Stubbe (lower left) with her first graduate students at the University of Wisconsin-Madison in 1981.

In all RNRs, thiyl radical formation is generated by an oxidant that resides in the same place in three-dimensional space within the α subunit. In the E. coli and human Ia RNRs, however, a redox inert tyrosine exists where the oxidant should be. The discovery in 1977 of an essential tyrosyl radical in the second subunit, β, in class Ia RNRs set the stage for understanding an unprecedented reaction in biology: an oxidation that occurs between the tyrosyl radical and a cysteine across two protein subunits, β and α, over more than 35 Å!

This distance is based on a docking model from the X-ray structures of each subunit and was proposed by U. Uhlin and H. Eklund (Nature 1994, DOI: 10.1038/370533a0). Oxidation over such a long distance requires a mechanism involving multiple protein radical intermediates and the coordination of electron and proton transfers at each step, all while avoiding self-destruction. Biophysical methods that have detected amino acid radical intermediates and a recent cryo-electron microscopy structure of an “active” RNR support this amazing model. These enzymes are proton-coupled electron transfer (PCET) machines. Not only is a metal cluster essential for generating the tyrosyl radical in subunit β, but it also plays an essential role in the radical transfer over 35 Å to initiate the chemistry of NDP reduction in subunit α.

Evidence to support this model has required experimental ingenuity because rate-limiting physical steps (or a step) mask both NDP reduction and radical transfer. To study this system, the following methods have allowed my group in collaboration with others to uncouple conformational gating, detect radical intermediates, and measure the kinetics of radical formation. The new tools to unravel the radical transfer process involve:

• Site-specific incorporation of unnatural tyrosine analogs with perturbed reduction potential and pKas, central to understanding PCET chemistry

• High-field and multifrequency paramagnetic resonance spectroscopies (EPR, ENDOR, PELDOR), which allow structural characterization of the radical intermediates and distance measurements between the radicals and protons

• Construction of phototriggered RNRs that uncouple conformational gating that, in conjunction with unnatural amino acids that have been site-specifically incorporated, allows triggering of the reaction with light on the nanosecond timescale and measurements of rate constants of 10x s–1 (x = 4 to 6)

• Mechanism-based inhibitors to unravel active-site chemistry and act as potential therapeutics

RNRs use exquisitely controlled nucleotide- and protein-radical chemistry to make the building blocks for DNA in all organisms. They also reveal the consequences of small perturbations of the protein or substrates that lead to radicals that are out of control. Examples of mechanism-based inhibitors of RNRs are 2′-substituted deoxynucleotides in which the 2′-hydroxyl group of the dNDPs is replaced with a fluoro, chloro, or difluoro group. Gemcitabine (which contains a difluoro group), used clinically in the treatment of pancreatic cancer, is an example (Proc. Natl. Acad. Sci. U.S.A. 2007, DOI: 10.1073/pnas.0706803104).

This scheme, depicting the proposed action in the active site of ribonucleotide reductase (in the α subunit), shows six intermediates, with the thiyl radical (top, panel 1) initiating the first step after being generated by the tyrosyl radical in the β subunit. Through hydrogen transfer and proton-coupled electron transfer steps, the hydroxyl group is removed, and the nucleotide is reduced. The deoxyribonucleotide produced (panel 6) is then released, the tyrosyl radical in β is regenerated, and the disulfide is reduced before the process begins with a new substrate.
Six-part scheme showing the reduction of ribonucleotides to deoxyribonucleotides.
This scheme, depicting the proposed action in the active site of ribonucleotide reductase (in the α subunit), shows six intermediates, with the thiyl radical (top, panel 1) initiating the first step after being generated by the tyrosyl radical in the β subunit. Through hydrogen transfer and proton-coupled electron transfer steps, the hydroxyl group is removed, and the nucleotide is reduced. The deoxyribonucleotide produced (panel 6) is then released, the tyrosyl radical in β is regenerated, and the disulfide is reduced before the process begins with a new substrate.

Nature has figured out how to harness the reactivity of radicals to carry out difficult chemistry with exquisite specificity. RNR is a paradigm for controlled radical chemistry. However, as with the Berkeley student protesters, it is all about homeostasis. As shown with gemcitabine, a small mechanistic perturbation results in inhibition of RNR and subsequent inhibition of DNA replication and repair.

My success in science has been related to the identification of important problems and their pursuit with intensity. The most rewarding part of my career has been training young scientists (graduate and undergraduate students and postdoctoral fellows) in the scientific method and the pursuit of truth. Experiments often lead in unanticipated directions. Rethinking, reformulating, and further experimenting lead to new models and, when we are lucky, to the thrill of discovery. Reading papers of my former coworkers often gives me a great sense of pride as an educator. Thank you for all your discoveries. I am grateful for my collaborators, as well as my coworkers, who have played an essential and central role in testing and reformulating new models. Thank you for sparking open and intense discussions and for making us collectively think. I am grateful to my families—my scientific family and my biological family—for the acceptance of my all-encompassing, often selfish, scientific intensity. So thank you, thank you, thank you so much.

While many have told me that they thought I would die with my boots on, that has never been my intention. I believe that the most creative ideas come from the young generation, unencumbered by past prejudices. Thus, I close with another Andrew Wyeth painting, titled Distant Thunder, and a poem—sung by guess who?

The line it is drawn
The curse it is cast
The slow one now
Will later be fast
As the present now
Will later be past
The order is rapidly fadin’.
And the first one now
Will later be last
For the times they are a-changin’.

—Bob Dylan, “The Times They Are A-Changin”.


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