Insights Gained by EPR Spectroscopy into the Composition and Function of the Mitochondrial Respiratory Chain

Abstract

Ever since Keilin’s fundamental discovery—or rediscovery—in the 1930s of the cytochromes (Keilin, 1966), which have been and still are considered by many to be the mainstays of the mitochondrial respiratory chain, optical spectroscopy has played a paramount role in this field of research. The past two or three decades have witnessed the introduction into biochemistry of additional and different forms of spectroscopy, using frequencies of the electromagnetic spectrum considerably higher or lower than traditional spectrophotometry, which of course often required radically different technologies. In its impact on our knowledge of what has been called the respiratory chain, one form of spectroscopy stands unequaled, viz. electron paramagnetic resonance (EPR) spectroscopy. This might have been expected, for EPR spectroscopy is the spectroscopic technique to directly look at unpaired electrons in organic molecules—e.g., cofactors such as flavin or ubiquinone (UQ)—and transition metal ions, which are electron carriers in oxidative enzymes. Fortunately, other virtues of the technique have joined this most important quality to make EPR spectroscopy the thus far most successful tool in advancing our knowledge far beyond the status as essentially set by Keilin’s pioneering work and by logical extensions thereof in the 1940s and 1950s. The virtues are as follows. (1) The sensitivity of EPR spectroscopy, when carried out at low temperatures, is sufficient to let us clearly recognize even in whole tissues practically all the electron carriers that have been identified by EPR in more concentrated, purified fractions. (2) While the sensitivity of EPR spectroscopy, in most instances, lies one or two orders of magnitude below that of spectrophotometry, its power of discrimination is far superior, particularly because effects of the intensity of the impinging radiation (saturation), observation temperature, and coupling of the observed paramagnet to other magnetic species can be exploited.