Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b(561) homologue

Candidate human tumour suppressor gene product, 101F6 protein, is a highly hydrophobic transmembrane protein and a member of cytochrome b(561) family. Purified 101F6 protein expressed in Pichia pastoris cells showed visible absorption spectra similar but distinct from those of cytochrome b(561). Haem content analysis indicated presence of two haems B per molecule. Midpoint potentials of the purified protein were found as +109 and +26 mV for two haems, slightly lower than those for bovine chromaffin granule or plant Zea mays cytochromes b(561). Electron paramagnetic resonance (EPR) spectra in oxidized state at 5 K showed only a highly anisotropic low-spin (HALS) signal at g(z) = 3.75. However, at 15 and 20 K, another HALS-type signal appeared at g(z) = 3.65 being overlapped with that of g(z) = 3.75. The rhombic EPR signal at g(z) = 3.16 previously seen in other cytochromes b(561) was not observed, suggesting distinct haem environments. Absence of the inhibition in the electron transfer from ascorbate by a treatment of 101F6 protein with diethylpyrocarbonate showed a remarkable contrast from those of other cytochromes b(561) where the 'concerted H(+)/e(-) transfer mechanism' at the cytosolic haem centre was blocked by specific Nε-carbethoxylation of haem-coordinating imidazole, suggesting that 101F6 protein might accept electrons via a mechanism distinct from other cytochromes b(561).