The scientists, a group from the California Institue of Technology and San Francisco's Department of Veterans Affairs Medical Center, solved the structure of fumarate reductase to a resolution of 3.3 Å by using multiple-wavelength anomalous diffraction (MAD). They took advantage of the tunability of ALS wiggler light, collecting data sets at three different wavelengths near the iron K edge. The final model was a good fit with the diffraction data (R_cryst = 22.2% and R_free = 29.2%), particularly for a membrane protein, as these are traditionally difficult to solve. The intensity of the light from Beamline 5.0.2 gave a higher signal than would have been possible at most other crystallography facilities.

The new structure shows how the four subunits of fumarate reductase fit together. The subunits include a flavoprotein, an iron-sulfur protein, and two membrane anchor subunits that associate with quinone molecules. These four subunits turned out to be arranged in the shape of the letter "q," where the flavoprotein and the iron-sulfur protein comprise the circle, and the membrane anchors constitute the stem. The whole structure is very modular, with the flavoprotein subunit associating only with the iron-sulfur subunit, and the iron-sulfur subunit in turn associated with the two membrane anchors. The q-shaped complexes were found in pairs. These were associated through their membrane-anchor regions, yielding a shape like the letters "qp." Although this structure gives the initial appearance of being a dimer, the lack of physiological evidence to support dimerism and the short length of the contact region make it unlikely.

So Who Cares about Breathing?

We all have an instinctive feel for the necessity of breathing, but how does that relate to respiration in bacteria? To understand its importance, you need to understand just what respiration is. To a biologist, it's the production of energy in cells. For aerobes, such as people, this process is very much dependent on oxygen. For anaerobic bacteria, it depends on other substances, depending on the kind of bacteria and their environment. One substance that bacteria can use in respiration is fumarate. In this way of life, fumarate reductase plays a starring role, catalyzing the final reaction that generates adenosine triphosphate (ATP), the currency of energy exchange within the cell. The same reaction happens in reverse in aerobic respiration, and it's catalyzed by succinate dehydrogenase. This reverse reaction shows up as a step in the Krebs cycle, which generates the molecules needed to produce ATP, and in the aerobic respiratory chain, where most of the ATP is actually produced. Though fumarate reductase and succinate dehydrogenase catalyze opposite reactions, they are very similar proteins. Thus, understanding the structure of one of them sheds light on the structure of the other and hence illuminates the processes of both aerobic and anaerobic respiration.

Since the crystals studied contained bound oxaloacetate, which inhibits fumarate reductase activity, the presence of the inhibitor near the flavoprotein subunit confirmed the flavoprotein as the active site for binding succinate or fumarate. The sequence of iron-sulfur clusters in the iron-sulfur protein subunit also confirmed previous studies, which predicted the 2Fe-2S moiety to be nearest the flavoprotein and the 3Fe-4S one to be nearest the membrane anchors. The membrane anchors were found to span a surprisingly long distance, putting the two quinones a physiologically unlikely 27 Å apart. Conducting electron transport over such a distance would be energetically difficult. The solution may be a cavity found nearly midway between the two quinone sites, serving as a way station only 13 Å from one quinone and 14 Å from the other.

Structure of the E. coli fumarate reductase respiratory complex.	Spatial relationships between cofactors in the complex.

The new information about fumarate reductase provides a framework for understanding the function of an entire family of proteins. The similarity of its subunits to other respiratory protein structures also gives intriguing clues to the functional origins of the protein. Together with recently solved structures within complexes III, IV, and V of the respiratory pathway, it gives a more complete picture of respiration, both with and without oxygen.

Research conducted by T.M. Iverson and D.C. Rees (California Institute of Technology) and C. Luna-Chavez and G. Cecchini (Department of Veterans Affairs Medical Center, San Francisco).

Research Funding: U.S. Department of Veterans Affairs, National Institutes of Health, National Science Foundation, and the Howard Hughes Medical Institute. Operation of the ALS is supported by the Division of Materials Sciences, U.S. Department of Energy.

Publication about this research: T.M. Iverson, C. Luna-Chavez, G. Cecchini, and D.C. Rees, "Structure of the Escherichia coli Fumarate Reductase Respiratory Complex," Science 284, 1961-1966 (1999).