The Ci/bH Moiety in the B6f Complex Studied by EPR: a Pair of Strongly Interacting Hemes

X-band EPR features in the region of 90-150 mT have previously been attributed to heme ci of the b6 complex [Zhang H, Primak A, Bowman MK, Kramer DM, Cramer WA (2004) Biochemistry 43:16329-16336] and interpreted as arising from a high-spin species. However, the complexity of the observed spectrum is rather untypical for high-spin hemes. In this work, we show that addition of the inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide largely simplifies heme ci's EPR properties. The spectrum in the presence of 2-n-nonyl-4-hydroxyquinoline N-oxide is demonstrated to be caused by a simple S = 5/2, rhombic species split by magnetic dipolar interaction (A(xx )= 7.5 mT) with neighboring heme bH. The large spacing of lines in the uninhibited system, by contrast, cannot be rationalized solely on the basis of magnetic dipolar coupling but is likely to encompass strong contributions from exchange interactions. The role of the H2O/OH- molecule bridging heme ci's Fe atom and heme bH's propionate side chain in mediating these interactions is discussed.