Investigation of the Mn Binding Site in Cytochrome c Oxidase from Paracoccus denitrificans by High-Frequency EPR

Cytochrome c oxidase contains a redox-inactive metal center of unknown function. It has been shown that for Paracoccus denitrificans, depending on growth conditions, Mn2+ ions can occupy the Mg2+ site in the functional protein. Differences between the 9.5 GHz X-band electron paramagnetic resonance (EPR) spectra of such Mn2+ preparations in oxidized and reduced state have previously been interpreted as being due to conformational changes. However, only minor differences have been found in analogous 34 GHz Q-band EPR experiments on Mn2+ containing cytochrome c oxidase from Rhodobacter sphaeroides. The new temperature-dependent X-, Q- and 95 GHz W-band EPR data from Paracoccus preparations shown here provide a consistent explanation of these contrasting former interpretations. The EPR spectra of oxidized samples reveal contributions of a dipolar interaction between the Mn2+ spin and the paramagnetic dinuclear CuA center. In reduced samples, the CuA center is diamagnetic, and thus, a pure Mn2+ signal is found. The zerofield parameters D and E are 145 ± 10 (120 ± 5) G and 28 ± 5 (22 ± 3) G in the fully oxidized (reduced) protein. From the evaluated dipolar coupling constant of 33.6 ± 1 G, an averaged distance between Mn2+ and the CuA center of 9.4 ± 0.2 Å was calculated, providing an independent confirmation of the distance calculated from the 2.7 Å X-ray structure.