Optical and Resonance Raman Spectroscopy of the Heme Groups of the Quinol-Oxidizing Cytochrome aa₃ of Bacillus subtilis

The cytochrome aa₃-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa₃-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CU_A center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The α-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa₃-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa₃-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa₃-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa₃-type oxidases. Raman modes arise at 194 and 214 cm⁻¹in aa₃-600, whereas a single band at about 214 cm⁻¹ is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa₃-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.