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

Abstract

We have designed andassembled 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-' gratings blazed at 400 nm), has excellent stray light rejection and optimum bandpass and resolution (5-12 cm-') for biological applications. It is demonstrated that a 600 cm-' 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 x lo4 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-' in the spectrum of ferrocytochrome c. Using the SIT detection system, the Raman excitation profile of a depolarized line at 750 cm-' 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.

Resonance Raman spectra of heme c and heme d1 in Pseudomonas cytochrome oxidase

“…Only in the 300-400 cm -L regions do significant qualitative differences appear. It is in this region that band splitting in cytochrome c has been reported to occur, presumably due to removal of axial degeneracy [8]. Differences among other cytochromes c are also evident in this region [9].…”

A model for the misfolded bis-His intermediate of cytochrome c: the 1–56 N-fragment

“…2). At neutral pH the RR spectra of cyt c are extremely rich, especially in the low-frequency region [19] due to the heme-protein interactions which result in a pronounced saddling distortion of the heme group in the native structure [20]. Nevertheless, its RR bands were completely assigned [21].…”

Resonance Raman investigation of dioxygen bonding in oxycobaltmyoglobin and oxycobalthemoglobin: structural implication of splittings of the bound O--O stretching vibration.

“…Raman spectra were obtained by using a highly sensitive multichannel Raman system, which has been described in detail elsewhere (25). The spectrometer consists of two 600 grooves per mm classically ruled gratings in additive dispersion, which has been demonstrated (20,26) to be well suited for resonance Raman spectroscopy of heme proteins.…”