Noninvasive Auto-Photoreduction Used as a Tool for Studying Structural Changes in Heme-Copper Oxidases by FTIR Spectroscopy

Abstract

We demonstrate an efficient Fourier transform infrared (FTIR) spectroscopic method, termed "auto-photoreduction," that uses anaerobic photo-induced internal electron transfer to monitor reaction-initiated changes of heme-copper oxidases. It can be applied without the use of either expensive electrochemical equipment, or caged compounds, which cause significant background signals. At high irradiation power, carbon monoxide is released from high-spin heme a of cytochrome c oxidase and heme o from cytochrome bo(3). Photochemistry is initiated at wavelengths <355 nm, and the photochemical action spectrum has a maximum of 290 nm for cytochrome bo(3), which is consistent with the possible intermediate involvement of tyrosinate or an activated state of tyrosine. We propose that the final electron donors are proton channel water molecules. In the pH range of 4-9, the noninvasive auto-photoreduction method yields highly reproducible FTIR redox difference spectra within a broad range, resolving a number of vibrational changes outside the amide I region (1600-1640 cm(-1)). Furthermore, it provides details of redox-induced changes in the spectral region between 1600 and 1100 cm(-1). The auto-photoreduction method should be universally applicable to heme proteins.

Photoreactions of Cytochrome c Oxidase

“…Table 1 shows that the quantum yields for heme A photoreduction in CcO become larger with increasing photon energy, consistent with earlier observations (25,30). The photoreaction appears to be essentially monophotonic, with no significant change in total quantum yield on change in excitation density, D (Table 1).…”

“…The data were consistent with light‐induced autoreduction of the enzyme, followed by oxidation of its two‐electron reduced form by dioxygen. Bettinger et al (30) recently investigated the anaerobic autophotoreduction of several oxidases, including bo 3 from E. coli and aa 3 from bovine heart and R. sphaeroides , with UV light. The photoreduction was initiated by irradiation at or below 355 nm; an action spectrum with a maximum at 290 nm was observed for the bo 3 enzyme.…”

“…Photoreduction can be accomplished in an otherwise closed system, eliminating the need for external reductants and electrochemical instrumentation (30). Hence, autophotoreduction is particularly useful for FTIR difference spectroscopy, which relies on a stable reference spectrum for detecting small changes in enzyme structure (30). By varying light intensity or duration of exposure, it is relatively simple to control the extent of reduction.…”

“…External, low potential reductants react with the enzyme to produce the fully reduced enzyme, as well as a mixture of intermediate, equilibrating oxidation states, depending on the number of reducing equivalents delivered to the enzyme (19,20). Similarly, irradiation of the oxidized enzyme with visible or UV light results in autophotoreduction (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), a process that is poorly understood (30,31).…”

Photobiochemical Production of Carbon Monoxide by Thermus thermophilus ba3‐Cytochrome c Oxidase

“…It should be noted that by using intense ultraviolet irradiation (308 nm) to E. coli bo 3 and Rhodobacter sphaeroides aa 3 oxidases, to monitor the auto-photoreduction process, a significant production of CO was reported. [28] Although our understanding of the light-induced electrontransfer mechanism is far from complete, we propose that Tyr237 which is cross-linked to the Cu B ligand His233 and hydrogen-bonded to the heme a 3 OH group (O-O distance at 2.52 ) may act as an electron donor in the CO release mechanism. The formation of the Tyr237 radical has been proposed to play a crucial role in the cleavage of the O=O bond during the reduction of O 2 to water.…”