Robert Kranz has devoted much of his career to understanding cytochrome c, one of the most interesting of a large group of biological molecules called the cytochromes.
Few people have heard of the cytochromes, much less of cytochrome c. But if these molecules were suddenly to stop working, we would fall over dead. The cyanide pill the trapped spy crushes between his teeth kills him because the cyanide binds to cytochrome, preventing it from doing its job.
When Kranz first turned his attention to these molecules, scientists thought there was only one system (or biological pathway) for making cytochrome c. Now, thanks largely to his work, we know there are three different pathways.
Why three? The pathways tell a fascinating story about hostile chemical environments and ingenuous biological strategies for avoiding or resisting them.
But three is also a lucky number. Many organisms that cause disease make cytochrome c by pathways different from ours, a difference we might be able to exploit in new antibiotics and other medicinal drugs.
Kranz, Ph.D., professor of biology in Arts & Sciences at Washington University in St. Louis, has been driven by curiosity and a desire to understand basic science, but medical applications naturally fall out of a deep understanding of a molecule as important to life as cytochrome c.
Too little iron
Cytochromes are part of a Rube Goldberg-like contraption in the mitochondrial or bacterial membrane that is called an electron transport chain. The chain, which consists of as many as 40 different proteins, wrings energy out of food by extracting electrons from sugars, siphoning off a bit of their energy and passing them along to the next molecule in the chain, which also siphons off a little energy, and so on.
Cytochrome c’s “active site,” the place where all the chemistry happens, is an iron atom held in a circlet of atoms like a gemstone in a setting. The circlet of atoms is called a heme group.
Most of the iron in our bodies is tucked inside heme groups that are themselves protected by larger proteins. “The iron’s usually chelated all up,” says Kranz. “And that’s probably by necessity.”
Why hide the iron?
One reason is that free iron is chemically active and could do enough damage to kill cells. Binding it to proteins allows cells to control its activity. Another is that bound iron is harder to steal. There is so little free (unbound) iron in the human body that micro-organisms trying to colonize us have evolved clever ways to expropriate our iron. Some send out small, iron-seeking compounds, called siderophores, that they then reabsorb to recover the iron.
Iron is that important.
Kranz studies cytochrome c, a particularly robust cytochrome that protects its heme by holding it in a tight chemical embrace.
Cytochrome c has to be tough because it sits on the outside of the membrane of a bacterium or mitochondrion where it is exposed to “environmental disasters such as acids, bases and oxidizers” says Kranz.
“So very early on—maybe three billion years ago—organisms evolved a way to make themselves more stable by putting the heme covalently onto the cytochrome protein,” he explains. (As bonds go, covalent bonds are strong ones.)
“Our studies address how different organisms do that,” Kranz says. In a 1998 review article Kranz and colleagues called the three known mechanisms systems I, II and III. System I includes nine proteins, system III has only one, and system II has an intermediate number.
People have the third pathway, System III, which consists of a single enzyme. But not just people. So do all invertebrates, all vertebrates and even yeast. (And if you’re wondering what that leaves, think plants and bacteria, among other things.)
We probably have System III because the chemical reactions important to us take place inside organelles, which are themselves inside cells. We can get away with a simple system because our organelles protect the heme from chemical assault until it is firmly strapped down to the cytochrome molecule, Kranz says.
System III was the first system to be discovered, in 1987. Scientists at the University of Rochester found a mutant yeast strain that couldn’t make cytochrome c and, comparing the mutant strain to normal strains, were able to identify the enzyme.
Another system discovered
Mutants also revealed the second biological pathway. This time it was mutants of Rhodobacter capsulatus, a bacterium isolated from ponds in Forest Park in St. Louis (among other locations), that has long served as a model organism in biology labs.
Rhodobacter can alter their metabolism to grow under a wide variety of conditions, both in the light and in the dark, and with and without oxygen. Because his mutants couldn’t grow in the light without oxygen, Kranz thought there was something wrong with the switch that turned their photosynthesizing machinery on and off. But the defects turned out instead to be in the machinery for making cytochrome c.
By 1989 his team had published a paper describing the first genes in a new cytochrome biosynthesis pathway, called System I. By the early 1990s the Kranz group and another in Switzerland had found all the genes in this pathway.
Fully nine proteins make up the elaborate cellular cytochrome assembly system in Rhodobacter capsulatus
A third system
System II, the last system to be found, was discovered in 1996 in the chlorophasts of an algae called Chlamydomonas by a UCLA group led by Sabeeha Merchant. Kranz’s team soon set to work to take this system apart and see how it worked.
In a mini-review published in 2002 Kranz’s team recommended Bordetella pertussis, the bacterium that causes whooping cough, as a model organism for System II. Like many bacteria it has several electron transport chains and, if one is disabled, it can switch to another.”You can’t work on an organism that dies if you delete the genes in a pathway,” Kranz explains. “That’s not a good model organism.”
Using a strain of pertussis from the medical school at Washington University they set out understand the cytochrome c biofactory by mutating it piece by piece.
“You know you have the gene for a particular protein in a pathway if you mutate that gene and the organisms can’t make cytochrome c’s,” Kranz says. “We made four separate mutants in four separate genes in pertussis,” he continues, “and none of the mutants could make cytochrome c. “
So those four genes were part of system II, but were they all of it or were other genes involved as well? Some scientists thought there were more.
To find out Kranz’s team pulled the kind of stunt that makes people regard molecular engineering as modern magic.
Escherichia coli, the scientists knew, normally uses system I, the complicated pathway, to make cytochrome c. Kranz’s group deleted all the System I genes in E. coli and spliced in what they supposed to be a full complement of System II genes taken from yet another bacterium, Helicobacter hepaticus, a bug that can cause hepatitis. When the recombinant E. coli started making cytochrome c, they knew they had the complete System II biofactory.
That result was featured in Molecular Microbiology in 2006.
At this point they were finally in a position to ask why there were three different systems. The obvious guess is that System I, which is found in Archae, ancient, single-celled organisms, is the oldest system. The other systems might have arisen as organism streamlined System I so they wouldn’t have to invest so much energy in building it.
Kranz didn’t think this was necessarily the case, however, because the three systems aren’t distributed across species in the right pattern.
“The energy-making organelles in our cells have System III, the simplest system,” he says. “But plant mitochondria still have System I, the most complex system. So do some protozoa. The question is why.”
His guess is that the cytochrome c biofactories in plants and protozoa are exposed to harsher chemical environments than are our biofactories. Because of their local chemical environment, they have trouble protecting heme and keeping the iron it shelters in a biologically useful form.
“Consider a plant,” he says. “When it is photosynthesizing, it releases bursts of oxygen and other nasty oxidizing molecules. At the same time it’s trying to take a reduced iron atom and attach it to a cytochrome.”
So one hypothesis is that System I—and probably also System II—survive because they cope better with harsh chemical environments.
But another hypothesis is that the most elaborate system survives because it can cope with low iron levels in the environment. Kranz’s team used two strains of engineered E. coli to test this idea. They spliced System I genes into one strain and System II genes into the other strain. When they gradually decreased the iron to which the organisms had access, the System II strain stopped making cytochrome c, but the System I strain was unfazed.
Work on cytochrome c biosynthesis continues in Kranz’s lab. (See related stories “Heme Channel Discovered” and “Ancient System Assembles Cytochrome C by Bucket Brigade”). In the meantime, however, those recombinant E. coli are turning out to be useful. Kranz has put them to work testing chemicals for antibiotic activity. The E. coli assay, which is fast and cheap, will be used to screen thousands of chemicals in a National Institutes of Health library of chemicals for the ability to kill System I organisms.
“If we find a chemical that poisons the System I pathway,” he says, “we could kill bacteria without harming ourselves,” Kranz says. In other words, Kranz’s devotion to basic science could lead to a whole new category of antibiotics.
When that day comes, Kranz’s lab will be the place to party.