Complete spectra of the far-red chemiluminescence of the oxygenase reaction of Mn²⁺-activated ribulose-bisphosphate carboxylase/oxygenase establish excited Mn²⁺ as the source

Chemiluminescence emitted by Mn²⁺-activated ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) while catalyzing oxygenation was analyzed to clarify the source of the emission. Using dual detectors capturing radiation over a wide range of visible and infrared wavelengths, we tested for radiation from singlet O₂ decay and found it to be essentially absent (less than 0.1% of the total luminescence intensity). Spectra were determined between 647 and 885 nm with a very sensitive, charge-coupled detector-based spectrograph to detect differences in the emission spectra between rubiscos from bacterial and higher plant sources. All Mn²⁺-activated rubiscos emitted a broad, smooth spectrum of chemiluminescence, unchanging as the reaction progressed. The spectra from higher plant rubiscos (spinach and both the wild type and an L335V mutant from tobacco), all exhibited maxima at about 800 nm. However, Mn²⁺-activated rubisco from the bacterium, Rhodospirillum rubrum, emitted at shorter wavelengths (760 nm peak), demonstrating host ligand-field influences arising from aminoacyl residue differences and/or conformational changes caused by the absence of small subunits. The findings provide strong evidence that the chemiluminescence arises from an excited state of the active-site Mn²⁺ that is produced during oxygenation. We propose that the Mn²⁺ becomes excited by a one-electron exchange mechanism of oxygenation that is not available to Mg²⁺-activated rubisco.