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DURHAM, N.C. - Science's understanding of the evolution and
role of hemoglobin, considered one of the most studied and
best-understood molecules in nature, is being rewritten with
the help of a common intestinal parasite that infects more than
1 billion people worldwide.

A team led by Dr. Jonathan Stamler, a Howard Hughes Medical
Institute investigator at Duke University Medical Center,
believes that the hemoglobin molecule found in the gut of
Ascaris lumbriocoides is a remnant of a crucial evolutionary
junction in which anaerobic life forms, like bacteria,
separated from newly emerging aerobic organisms, such as
humans. The worm, in short, reveals that hemoglobin evolved
first and foremost to handle the molecule nitric oxide (NO)
rather than oxygen, as scientists thought, and tells the tale
of when hemoglobin ceased being a "consumer" of oxygen and
became a "carrier" of oxygen, Stamler said.

In a report published in the Sept. 30 issue of the journal
Nature, Stamler and
collaborators from Washington University in St. Louis provide
biochemical proof to support this conclusion. They show that
the worm actually consumes oxygen - which it finds toxic - with
the help of NO. The discovery may yield new therapies for
diseases such as cancer, in which starving tumors of oxygen is
a major therapeutic focus, the researchers said.

The research was supported by grants from the National
Institutes of Health and the Howard Hughes Medical Institute
(HHMI).

"Both structurally and functionally, Ascaris hemoglobin is a
link between bacterial hemoglobin and mammalian hemoglobin,"
said Washington University cell biologist Dena Minning, who,
along with Duke's Andrew Gow, are co-first authors of the
paper.

"Hemoglobin in bacteria is used as an enzyme to destroy NO -
the 'primordial gas' which evolved before there was oxygen and
is toxic in high amounts - while in mammals, hemoglobin carries
both oxygen and NO, using the NO to ensure oxygen delivery by
dilating blood vessels," Minning said. "But in the Ascaris
worm, NO is used to remove oxygen. Thus Ascaris hemoglobin is
for the first time regulating the oxygen in its environment,
although in this case it is getting rid of it.

"From the standpoint of hemoglobin biology, this finding
represents a new function and a novel mechanism for Ascaris
hemoglobin," she said. "In bacteria, hemoglobin's function is
detoxification, while in mammals, its function is respiration.
In Ascaris, at the evolutionary divide, we see the evolution of
the 'functional switch' - respiratory function (control of
oxygen) and the detoxification function (removal of oxygen) are
the same. And all is controlled by NO."

Minning's work in Ascaris biology was conducted in the
laboratory of Dr. Daniel Goldberg, a Washington University
microbiologist and HHMI investigator.

The keys to hemoglobin's evolution, the researchers believe,
are the chemical reactions that take place between hemoglobin,
oxygen and NO, a ubiquitous chemical involved in many life
processes. These findings suggest that contrary to commonly
held beliefs, hemoglobin has evolved over millions of years in
response to NO, and not oxygen.

"More than a billion years ago, the atmosphere on Earth
contained NO, and not oxygen," Stamler explained. "So the early
development of hemoglobin in bacteria and other microbes could
not have been for the delivery of oxygen, but instead was for
the detoxification of NO. On the evolutionary tree, the Ascaris
worm sits right at the point where bacteria branches off one
way and man in another," he said.

"We've known that hemoglobin in Ascaris binds oxygen 25,000
times more tightly than human hemoglobin - the tightest that
oxygen is held by any molecule," Minning said. "It's been a
mystery why a hemoglobin that is supposed to deliver oxygen
would bind it so tightly that oxygen could never be released, a
mystery that has baffled scientists for more than 50 years. Now
we know that this oxygen can be removed through a novel
enzymatic reaction with NO."

"In the beginning, oxygen was toxic to early plants and
worms and even late bacteria," Stamler said. "Early hemoglobin
would bind tightly to the oxygen and consume it, acting as a
detoxifier. However, in man, hemoglobin is the molecule that
carries oxygen. We know what the earliest hemoglobin did, and
we know what it does now in man.

"The Ascaris hemoglobin, in the presence of NO, consumes
oxygen, creating the hypoxic, or oxygen-free environment, which
it needs to live," Stamler said. "By doing so, the Ascaris
hemoglobin is really acting like a new enzyme, or a
deoxygenase, using NO to detoxify the oxygen around it."

It appears that structural differences have evolved in the
Ascaris and human hemoglobin molecules that account for their
different capabilities. The actual location on the molecule
where the chemical reactions take place differ in the two
hemoglobins, but they both employ a residue called cysteine
that enables hemoglobin to actively use NO.

"In the Ascaris hemoglobin, the cysteine carrying NO is at
the front, next to the oxygen, which leads to deoxygenation;
and in humans, it is located at the back, away from the
oxygen," Stamler said. "Since the gases are carried on opposite
sides of the molecule, they do not react with each other,
allowing for the release of NO and transport of oxygen. The
primordial function of detoxification has transformed into a
respiratory function in man."

While it appears that Ascaris hemoglobin is primarily
involved in the canceling out of oxygen, the Duke and
Washington University research also points to another function
- protecting the worm from NO that is present in the host gut
or produced by the host's immune system.

"This ability to metabolize NO is very similar to the
hemoglobin of early bacteria, but the Ascaris may not be able
to do it quite as efficiently, and does so through a different
chemical reaction," Stamler said.

The scientific world is beginning to recognize the
importance of NO in the evolution and functions of hemoglobin.
In a commentary in the Aug. 31 issue of the Proceedings of the National Academy of
Sciences, Drs. Steven Gross and Paul Lane of Cornell
commented on the new scientific view brought about in large
part by a series of publications over the past three years from
Stamler and his colleagues. These articles have been laying the
groundwork for challenging accepted beliefs about hemoglobin
and have revealed entirely new functions.

"This view would be consistent with the evolutionary
appearance of simple bacterial hemoglobin at a time when the
Earth's early atmosphere was anoxic, but perhaps
life-threatening in its NO content," Gross and Lane wrote.
"Thus, ancestral hemoglobin may have initially functioned to
detoxify NO and subsequently evolved toward a molecule that is
optimized for oxygen delivery, permitting the evolution of
large multicellular life forms. If so, we may owe our very
existence to evolutionary pressure imposed by an NO-rich
environment."

These new insights into hemoglobin and NO could have
profound effects on the development of drugs or compounds used
in a wide variety of medical conditions, including, heart
disease, shock, cancer and arthritis, Stamler said.