Resonance Raman spectroscopy of heme proteins with intensified vidicon detectors: Studies of low frequency modes and excitation profiles in cytochrome c and hemoglobin

Abstract

AbstractWe have designed and assembled a sensitive vidicon Raman spectrometer which is shown to be well suited for the studies of heme proteins in both Soret (near‐ultraviolet) and Q (visible) band regions. This system, employing a dry ice‐cooled silicon intensified target (SIT) detector and an additive dispersion double monochromator (two 600 g mm−1 gratings blazed at 400 nm), has excellent stray light rejection and optimum bandpass and resolution (5–12 cm−1) for biological applications. It is demonstrated that a 600 cm−1 wide section of the low frequency Raman spectrum of ferrocytochrome c excited at 413.1 nm (70 mW) can be obtained in 30 ms, which is ˜ 2×104 times faster than that required by the conventional scanning techniques. Our multichannel Raman system has an enhanced detection capability which enables us to identify at least 41 weak Raman lines below 850 cm−1 in the spectrum of ferrocytochrome c. Using the SIT detection system, the Raman excitation profile of a depolarized line at 750 cm−1 in Fe(II) cytochrome c has been constructed and compared with that reported for Ni etioporphyrin. Instead of strong 0–0 Raman intensity, the cytochrome c profile exhibits weaker 0–0 intensity, indicative of the absence of the Jahn‐Teller effect. It is further demonstrated that the Soret‐excited Raman spectrum of oxyhemoglobin can be readily obtained without photodissociation.