Human hemoglobin is a tetramer consisting of two α and two β subunits. Each subunit contains one identical ferrous heme group that can reversibly bind one ligand such as carbon monoxide (CO). Determining CO dynamics in hemoglobin subunits is essential for gaining insight into the transport of small molecules in physiological systems. Here, we use picosecond to millisecond transient mid-infrared (mid-IR) spectroscopy to study the photoinduced dynamics of CO in isolated hemoglobin subunits. Photoinduced absorption changes of the isolated carbonmonoxy hemoglobin subunits were measured after photoexcitation at 543 nm into the Q bands of the heme moiety. Time-resolved spectra in the mid-IR region were measured on the ULTRA apparatus at the Central Laser Facility (Didcot, UK). All the experiments were performed in 50 mM Tris buffer, pD 8.2, at 19°C.

The time evolution of the vibrational spectra of the coordinated and as photodissociated CO molecules was monitored in the spectral range between 1900 and 2180 cm^-1. The mid-IR spectrum of the liganded subunits shows discrete CO stretch bands, denoted A₀ (~1,968 cm^–1) and A₁ (~1,950 cm^–1). The distinct stretch bands for CO photolyzed and temporarily trapped in the protein matrix are detected in the region of 2,090–2,160 cm^–1. The measured transient mid-IR spectra were analyzed using singular value decomposition and maximum entropy method analysis. We succeeded in following the evolution of CO in hemoglobin subunits. The kinetic model, describing both the photodissociation and subsequent rebinding of CO, is introduced and discussed.