Profile of Michael A. Marletta

For more than three decades, Michael Marletta has traveled a serpentine road, working to elucidate a molecule whose importance to human physiology is as well recognized as his own contributions to his field. Named “Molecule of the Year” in 1992 by Science, nitric oxide, a noxious atmospheric gas, plays a pivotal role in biological functions as diverse as forming long-term memory and maintaining penile erections (1). Marletta, who was elected to the National Academy of Sciences in 2006, has shed light on the biochemistry of nitric oxide and the cellular machinery through which the molecule performs its functions.

Michael A. Marletta.

Marletta's work on nitric oxide not only led to a fundamental understanding of enzyme reaction mechanisms, but it has informed the development of treatments. For example, he showed that substrate lookalikes, called arginine analogs, block the activity of the enzyme nitric oxide synthase. The analogs help curb the overproduction of nitric oxide, preventing vasodilation and the precipitous drop in blood pressure experienced by patients suffering from septic shock (2). His discovery helped other researchers develop treatments for septic shock.

Marletta's scientific foray began in California, where he obtained his doctorate in 1978 from the University of California, San Francisco (UCSF). That journey, with stops in Massachusetts and Michigan, has led him back to California, where he is now the Aldo DeBenedictis Distinguished Professor of Chemistry and a professor of biochemistry and molecular biology at the University of California, Berkeley.

His love of science is deep-seated. Born in Rochester, NY, to parents of Italian ancestry, Marletta says the space race of the late 1950s spurred his scientific temperament. As a 6-year-old space buff, Marletta recalls watching Sputnik 1 fly overhead on a cold October night in 1957 in Rochester. “I was completely fascinated by the idea that a satellite launched by humans was orbiting the earth,” he says. That Christmas, Marletta asked his father for a telescope and spent many a winter's night stargazing. Over the next few years, his interest turned from astronomical to biological; he peered at living organisms procured from the neighborhood pond through a microscope.

His Italian heritage largely cultivated his interest in chemistry. “The Italians’ love for food was certainly prevalent in my house. My father worked at the R.T. French Company [a manufacturer of condiments, such as French's mustard]. He would come home at night, smelling like a spice mill. I can remember reading books in the library about the constituents of food, and I realized that foods smelled and tasted the way they did because of the molecules in them,” Marletta says.

He spent the next few years tinkering with chemicals in his basement laboratory. “In those days you could buy chemicals from drugstores. I had a paper route; I had money,” he chuckles. “And there was a library with books to tell you what to mix.” By age 10, Marletta knew he wanted to be a chemist.

“Biology Is for Wusses.”

Neither of Marletta's parents attended college, but both supported his interest in science. “My mother was a strong, encouraging force. I can still hear her telling me that anything was possible,” he says. During the fall of 1969, he enrolled at the State University of New York College at Fredonia to study chemistry. Between his sophomore and junior years, Marletta took a general biology course at the University of Rochester while working a summer job on campus. “Frankly, I remember thinking biology is for wusses. I figured if I got biology out of the way, I could have more time to take advanced chemistry courses,” he recalls. However, the course helped him realize that nature's ability to do chemistry surpassed that of chemists. “It's almost unbelievable how nature does intracellular chemistry largely in water, largely at a pH of 7.5, and mostly at 37 degrees Celsius. Chemists would consider those conditions about the worst to work with,” he says. Marletta's research during the next three decades focused on the interface between chemistry and biology.

Because of this work, he refuses to be typecast. “At one time in my career, I called myself a biochemist. Now, I call myself a chemical biologist, but I continue to do what I've always done—enzymology,” he says.

When it came time to pick a Ph.D. program, Marletta was faced with a dearth of choices because there were not many chemistry departments during the early 1970s that were serious about biology. However, a fortuitous tip from a professor at Fredonia, who had heard about a program in pharmaceutical chemistry at UCSF, pointed the way to Marletta's graduate studies.

His doctoral project in the laboratory of UCSF biochemist George Kenyon, now at the University of Michigan at Ann Arbor, was to investigate the mechanism of action of creatine kinase, an enzyme found abundantly in muscle and brain tissues, where it maintains constant levels of ATP, the cellular energy currency, by reversibly catalyzing the transfer of a phosphoryl group from ATP to the organic acid creatine. “It was a perfect project for me because it was my first experience in mechanistic enzymology,” Marletta says.

Marletta fashioned a substrate lookalike for rabbit skeletal muscle creatine kinase, called epoxycreatine, to probe the enzyme's active site (3). Until then, little structural information about the enzyme had been reported, and the enzyme's catalytic site had remained a black box. By binding specifically to the enzyme's active site and blocking its action, epoxycreatine helped explain the mechanism and kinetics of creatine kinase and the bioenergetics of ATP use in muscles. Additional studies using epoxycreatine by members of Kenyon's laboratory showed that a cysteine residue at position 282 of the enzyme's amino acid sequence was likely crucial to catalysis (4). Other researchers have since solved the enzyme's crystal structure (5).

Marletta's doctoral work on enzymes was not the only factor that cemented his long-term research interest. For his postdoctoral training between 1978 and 1980, Marletta worked with enzymologist Christopher Walsh at the Massachusetts Institute of Technology (MIT) on developing fluorinated analogs to study the mechanism of enzyme catalysis. The training turned into an assistant professorship for Marletta at MIT that lasted 6 years. “During my first year in Walsh's lab, I managed to get a couple of papers published, so I decided to test the job market,” he says. Fortunately, MIT's nutrition department was being restructured into the now-defunct applied biological sciences department into which Marletta was hired as an enzymologist for the toxicology program.

The Nitric Oxide Narrative

At MIT, he met toxicologist Steven Tannenbaum, whose work led to Marletta's research on nitric oxide. Tannenbaum studied how carcinogenic nitrosamines are made by the human body from nitrates found in drinking water and vegetables. Bacteria in saliva were known to convert nitrates into nitrites, which were then transformed into nitrosamines by intestinal bacteria. Tannenbaum's discovery that there were surprisingly high amounts of nitrates in the urine of experimental subjects fed a low-nitrate diet led him to hypothesize about and show the existence of a novel metabolic pathway for nitrate biosynthesis in humans (6). “Steve convinced me that mammals were making nitrate, and because I was a biochemist, I had to figure out where it was coming from,” Marletta says.

A serendipitous discovery by Tannenbaum's group pointed to the immune system as a source of nitrates: One of the experimental subjects, who had intestinal flu, was excreting whopping amounts of nitrate in her urine, suggesting that the source of the nitrate was either the pathogen or the immune system. Tannenbaum ruled out the pathogen and showed that rats injected with bacterial lipopolysaccharide to stimulate the immune system excreted large amounts of nitrates in their urine (7, 8). “That was a beautiful set of experiments that led me to ask what cells in the immune system were making the nitrate,” Marletta says.

Using biochemical methods, Marletta demonstrated that activated macrophages produced nitrates and were capable of forming nitrosamines under physiological conditions (9). “We figured out later that macrophages were converting arginine into citrulline, nitrite, and nitrate,” he says.

Despite those important findings, Marletta failed to obtain tenure at MIT. “MIT is a tough place, and the nitric oxide story wasn't fully developed until we moved to Michigan. At the time, we didn't know what tied the ability of macrophages to produce nitrate with their ability to kill invaders,” Marletta recalls. “I decided to take my chances and continue to pursue that line of research rather than something more predictable that might have gotten me tenure at MIT.”

At the University of Michigan at Ann Arbor, where he was a faculty member for 14 years, Marletta pursued that high-stakes research, which paid off in a big way. In 1980, pharmacologist Robert Furchgott found that endothelial cells lining blood vessels produced a chemical compound, called endothelium-derived relaxing factor (EDRF), which made the smooth muscles of vessel walls relax, thereby causing vasodilation (10). Extending Furchgott's findings, biochemist Salvador Moncada at University

Nature walks a tricky tightrope between toxicity and function.

College London and researchers elsewhere showed that EDRF was nitric oxide, a discovery with implications for the development of blood pressure drugs (11, 12).

“Moncada's Nature paper showing that EDRF was NO tipped me off,” Marletta says. In a 1998 landmark Biochemistry paper, Marletta showed that macrophages produce nitric oxide from arginine through a biochemical pathway similar to the one used by endothelial cells for vasodilation (13). The finding suggested that nitric oxide helped macrophages kill pathogens. “We found that nitric oxide was the intermediate between arginine and nitrate during nitrate biosynthesis in macrophages.” That discovery helped establish the role of nitric oxide in immunity, in addition to its known roles in vasodilation and neurotransmission.

“Here's an extremely toxic molecule that the immune system uses to kill, but which the body also uses to dilate blood vessels and to help neurons talk to each other. It's stunning, and that's why it captured a lot of attention,” Marletta says. Nature walks a tricky tightrope between toxicity and function by deploying no more than nanomolar to picomolar concentrations of nitric oxide, which usually decomposes once its work is done and before it can wreak havoc.

In 1995, Marletta won a MacArthur Foundation fellowship for his contributions to our understanding of the biochemistry of nitric oxide. Marletta was among the first to show that nitric oxide synthase, the enzyme that makes nitric oxide in macrophages, is a heme-containing protein that resembles cytochrome P450 and requires oxygen, NADPH, and tetrahydrobiopterin as cofactors for catalysis (14). “The enzyme catalyzes one of the most complicated redox transformations known, and that's what drew me to it. I've always been interested in enzyme catalysis,” he says.

This fact is finely illustrated by the slew of studies on nitric oxide synthase that Marletta published while at the University of Michigan, including reports on the enzyme's structure, catalytic mechanism, regulation, and inhibition (2, 15–18).

A New Direction

“I've always picked projects by looking for fundamental science with implications for human health and disease,” Marletta says. Thus, he turned his attention in 1995 to the biochemistry of malaria, a disease that kills nearly 2 million people each year. “My wife and I had our only child two months before I won the fellowship. I asked myself what new project I should work on. Seventy percent of those dying from malaria are under the age of 10, so I decided to study the biochemistry of the parasite,” he says.

The malarial parasite Plasmodium falciparum degrades the hemoglobin in host erythrocytes for its nutrition. The parasite feeds on the amino acids released from the globin; however, free heme's intracellular reactivity is toxic to the parasite. To counter heme's toxicity, a parasite protein called HRP-2 tucks heme into a complex called hemozoin. Marletta used spectroscopy to study the heme-binding site of HRP-2 and the molecular nature of the heme–HRP2 interaction (19). In another study with UCSF molecular biologist Joseph DeRisi and others, Marletta showed that a highly conserved, five-amino acid signal sequence in the N terminus of HRP2, called PEXEL—Plasmodium export element—undergoes enzymatic cleavage and acetylation in the parasite's endoplasmic reticulum before the parasite exports the protein to human erythrocytes (20).

Chasing Fresh Challenges

Unwilling to be tethered to the present and eager for fresh challenges, Marletta moved to the University of California, Berkeley in 2001. “I wanted a change and a new environment. I also longed to be back on the West Coast where I was once a graduate student. All of that aligned perfectly with my new position at Berkeley,” he says.

At Berkeley, Marletta's research has focused on understanding how cells distinguish nitric oxide from chemically similar oxygen, among other aspects of nitric oxide biology. Given that cells contain more oxygen than nitric oxide, it was long unknown how trace amounts of nitric oxide elicit specific cellular responses amid a sea of oxygen molecules. Nitric oxide performs its physiological role through an enzyme called soluble guanylate cyclase (sGC), which makes the signaling second messenger cGMP (21). cGMP, in turn, switches on a cascade of signals that leads to smooth muscle relaxation and vasodilation. Although nitric oxide toggles sGC between its active and inactive states, its mechanism is largely a mystery.

Marletta found that the part of the enzyme that binds nitric oxide contains a heme-binding region called heme-NO and oxygen-binding (H-NOX) domain. Together with Berkeley structural biologist John Kuriyan, Marletta solved the crystal structure of a similar H-NOX domain from a signaling protein in anaerobic bacteria. The structure, published in a well-cited PNAS article in 2004, provided the clue to how cells distinguish oxygen and nitric oxide (22). Marletta showed that specific amino acid residues in the H-NOX domain in sGC prevent the enzyme's heme from binding oxygen while allowing it to bind nitric oxide. Without this structural rejiggering, heme would bind oxygen, as it does with the oxygen-toting blood protein hemoglobin.

Marletta has studied the H-NOX domain in bacteria, worms, and flies, where proteins containing the domain selectively bind oxygen or nitric oxide for specific cellular functions. “It turns out that the domain can be a freestanding signaling protein in prokaryotes or fused to a larger protein, such as sGC,” he says. Pathogenic bacteria such as Vibrio cholera use the domain to bind small amounts of nitric oxide, an ability that likely evolved as an evasive strategy to neutralize the nitric oxide produced by macrophages (23). In collaboration with Rockefeller University neuroscientist Cornelia Bargmann, Marletta found that the nematode Caenorhabditis elegans harbors a homolog of sGC in specialized oxygen-sensing cells that the worm uses to detect oxygen levels in its environment and to regulate its feeding behavior. Unlike the H-NOX domain of sGC, the heme-binding domain of the worm homolog preferentially binds oxygen (24).

Years of work on the H-NOX domain resulted in Omniox, Inc., a California-based biotechnology company that Marletta cofounded in 2006 with his former graduate students Stephen Cary and Jonathan Winger. The company devises novel ways based on the H-NOX domain to selectively deliver oxygen or nitric oxide to tissues, an application with clinical implications for cancer, surgery, and cardiovascular diseases.

Marletta's contribution to nitric oxide biology earned him membership in the Institute of Medicine in 1999 and in the National Academy of Sciences in 2006. “I was excited when the IOM recognized our accomplishments and their impact on medicine. But, as a scientist, I must say that being elected to the NAS is what I am still excited about,” he says.

“We're going to continue to probe the expanding landscape of nitric oxide biology in eukaryotes and chart nitric oxide signaling pathways in pathogens,” Marletta says. That effort, he believes, could lead to treatments for an array of ailments, including rheumatoid arthritis, inflammatory bowel disease, and infectious diseases.