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No. 4, December 2007
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Tiny motors and hot bacteria
Tokyo Tech professor corroborates historic breakthroughs in biochemistry
The Tokyo Institute of Technology's Professor Masasuke Yoshida has helped elucidate the structure and the functioning of nature's smallest motor. Yoshida verified a crucial facet of the enzymatic mechanism hypothesized by the American biochemist Paul Boyer for the synthesis of adenosine triphosphate (ATP)—the universal energy carrier in cells. Previously, Yoshida participated in a group that helped verify the chemiosmotic coupling mechanism hypothesized by the British biochemist Peter Mitchell for ATP synthesis. For the discoveries corroborated by Yoshida, Mitchell received the Nobel Prize in Chemistry in 1978, and Boyer shared half of that prize with the British biochemist John Walker in 1997.

Boyer had hypothesized an enzymatic mechanism of ATP synthase in which a gamma subunit turns as an asymmetrical axle inside a cylinder formed by three alpha and three beta units. Scientists had struggled, however, to verify that rotation. Yoshida devised an innovative experiment to visualize the rotation in the F1 part of ATP synthase. Here is how the Royal Swedish Academy of Sciences described the achievement by Yoshida's research group in announcing the 1997 Nobel Prize in Chemistry:

They attached a fiber of the muscle protein actin to the gamma subunit, and the beta subunits were attached to the substratum. Depending on the ATP concentration in the surrounding liquid it was possible to show under a microscope how the actin fiber rotated at increasing speed with increasing ATP concentration.

"People resisted the notion of a rotational mechanism in synthase," recalls Yoshida, "and for good reason. We find lots of linear motors in cells, such as myosin, kinesin, and RNA polymerase, which operate on filamentous structures. But scientists have identified and investigated only one rotary motor, the bacterial flagellum. And whereas the flagellum is 50 nanometers in diameter, the rotary motor hypothesized by Boyer for ATP synthesis is only 10 nanometers in diameter. ATP synthase's rotational mechanism is the smallest motor in all of nature!"

The actin filament that Yoshida's group attached to the gamma subunit constituted the load on the motor. Correlating the angular velocity and the load allowed for accurately estimating the amount of work entailed in each 120-degree step of rotation. Yoshida determined that the frictional torque per step closely matched the free hydrolytic energy per ATP molecule in a cell. In other words, the motor's energy efficiency is uncannily high: nearly 100%.

ATP synthase1
Mitochondria burn hydrogen through respiration and emit hydrogen ions (the tiny blue spheres in the graphic). Differing concentrations of protons arise on opposite sides of the mitochondrial membrane, resulting in an electrochemical potential difference. That potential drives the "motors" of the ATP synthase.
Convincing the skeptics

ATP, discovered by the German chemist Karl Lohmann in 1929, is the energy carrier for all life on earth. In the 1960s, scientists regarded ATP as the product of substrate-level phosphorylation inside the mitochondria. Mitchell's chemiosmotic hypothesis rejected that assumption and proposed that differing concentrations of hydrogen ions on either side of the mitochondrial membrane drive the synthesis of ATP.

The scientific community was generally skeptical of the mechanism proposed by Mitchell. His hypothesis gradually gained acceptance, however, as research demonstrated its validity. A group in which Yoshida participated demonstrated the role of chemiosmosis in ATP synthesis by means of a highly simplified structure. They achieved ATP synthesis with vesicles reconstituted from purified ATP synthase and from phospholipids.

"Finding an adequate source of ATP-generating enzyme was a big step in our research," recalls Yoshida. "Scientists were using enzymes from cows and other animals, but the protein was extremely fragile and difficult to work with. The technique for isolating the enzyme involved rendering the cell membrane soluble, but that tended to damage the enzyme protein. We overcame that problem by using themophilic bacteria as our enzyme source. ATP and the enzyme that creates it are basically common to all organisms, so bacteria are as good a source as any."

Yoshida found his thermophilic bacteria at an ever-so-Japanese location: hot springs. Like most of his compatriots, Yoshida is extremely fond of lounging in spas. He reasoned that bacteria hardy enough to flourish in the hot springs would yield robust enzyme protein. His hunch proved correct and provided his research group with access to an abundant supply of raw material.

ATP synthase2
At the top is a computer simulation of the rotation of the ATP synthase. The yellow rod is a protein fiber Yoshida's group attached to the gamma subunit to monitor the rotation. The group fitted the fiber with a fluorescent marker to maximize its visibility. Below the computer graphic is a sequence of micrographs from a video of the turning fiber taken by Yoshida's group.
Unleashing the imagination

Teamwork is a high priority for Yoshida, and he readily cites the contributions by colleagues. He emphasizes the roles of Yasuo Kagawa and Nobuhito Sone, both at Jichi Medical College (now Jichi Medical University) at that time, in elucidating Mitchell's chemiosmotic coupling mechanism. And he names Kazuhiko Kinoshita, at Waseda University, as a crucial contributor in elucidating Boyer's enzymatic mechanism of ATP synthesis.

After earning a doctorate in biochemistry from the University of Tokyo in 1972, Yoshida lectured at Jichi Medical University, north of Tokyo, and served for two years as a visiting researcher at the University of California at San Diego. He has been at Tokyo Tech since 1985, where he heads the Chemical Resources Laboratory.

Yoshida speaks enthusiastically of the daring and counterintuitive insights by Mitchell and Boyer. He remains excited about the role he has played in verifying their discoveries. And he encourages his students to be bold in their thinking and in their research. Yoshida worries that the competition for research funding dissuades scientists from exploring ideas of uncertain feasibility but of potentially important ramifications. "Feasibility is important," he acknowledges, "but we also need to unleash our imaginations."

Pull quote_Yoshida

Masasuke Yoshida

Controlling the motor

Yoshida has continued to elucidate important aspects of the synthesis and hydrolysis of ATP. He is especially interested in how organisms suppress the reverse reaction of ATP synthase to prevent the wasteful consumption of ATP.

Multiple research groups, including Yoshida's, have long since demonstrated that the ε subunit in bacterial ATP synthase figures prominently in the suppression function. Yoshida's group has further discovered that the ε subunit alternates between two contrasting shapes: spherical and extended. That alternation appears to affect the speed of the motor and thereby regulate the pace of ATP synthesis.

More recently, Yoshida's group has determined that the spherical ε subunit binds directly with ATP, and the group has determined the structure of the resultant crystal. Yoshida is investigating the ε subunit's presumed role as a sensor for monitoring and regulating the intracellular concentration of ATP.

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