Performance characteristics are described for a kilohertz solid-state laser source for resonance Raman spectroscopy. Narrow line excitation in the 205–230 nm region is provided by quadrupling a titanium: sapphire laser which is pumped by the second harmonic of a Q-switched YLF laser. The combination of tunability, narrow line, high average power, and good stability makes this laser suitable for ultraviolet resonance Raman (UVRR) applications. UVRR spectra of hemoglobin, excited at 229 nm, are as high in quality as those produced by continuous-wave intracavity frequency-doubled Ar+ laser excitation. Raman excitation profiles are reported for aromatic acids in aqueous solution and in hemoglobin.

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New UV resonance Raman (UVRR) data provide convincing evidence that a characteristic 1511 cm(-1) band in the T - R difference spectra of hemoglobin is due to the overtone of the Trp W18 fundamental at 756 cm(-1). Measured isotope shifts for 2-H and 15-N substitution at the indole NH group are twice as large for the 1511 cm(-1) band as for W18, and the 1511 cm(-1) intensity scales with that of W18 in the difference spectrum. Moreover, the UVRR excitation profile of the 1511 cm(-1) band tracks that of another tryptophan band, W16. Both are redshifted in hemoglobin, relative to aqueous tryptophan, reflecting H bonding within a hydrophobic environment in the protein. The 2xW18 assignment had been thrown into question by the observation of remnant 1511 cm(-1) intensity in the T - R spectra of hemoglobin labeled with tryptophan-d(5), a substitution that shifts W18 over 50 cm(-1). However, reexamination of the data suggests that this remnant intensity may result from a subtraction artifact arising from the downshift of another difference band, W3, from 1549 cm(-1) in unlabeled protein to 1522 cm(-1) in labeled protein. Restoration of the 2xW18 assignment establishes that the 1511 cm(-1) difference band, which is a useful indicator of the extent of T-state formation in hemoglobin, arises from the same residue, Trpbeta37, that gives rise to essentially all of the T - R signal from tryptophan.

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Recent studies of Cu, Zn superoxide dismutase, and of zinc-finger peptides have established that histidine ligands can be detected in ultraviolet resonance Raman (UVRR) spectra, following NH/D exchange of the imidazole. UVRR spectroscopy therefore offers promise for monitoring histidine ligation in heme proteins. In this work, we characterize heme-bound histidine UVRR bands for N-acetyl-microperoxidase-8 (MP-8) and microperoxidase-11 (MP-11), and also for hemoglobin (Hb). The Hb UVRR spectra are dominated by tyrosine and tryptophan contributions, but a band appears at 1340 cm -1 in D 2 O solution, which is assigned to a mode of Fe-bound imidazole. This band shifted 24 cm -1 in protein which was labeled with 15 N via expression of the Hb gene in E. coli grown on 15 NH 4 + . In MP-11, the position of this band is insensitive to ligation or oxidation state changes, but it is 2 cm -1 lower in deoxyHb than in the CO adduct. This shift may reflect mechanical forces on the proximal histidine in the T state, and/or changes in its H-bonding.

Time-resolved FTIR spectra are reported for the photocycle of carbonmonoxy hemoglobin, under saturating photolysis conditions, which are sufficient to drive the R-T allosteric transition. Direct evidence for this transition was provided by the microsecond time scale evolution of an 1857 cm -1 bisignate difference band (cysteine S-H stretching), which is a marker of the T state. The time course of the strong 1951 cm -1 band of bound CO showed the expected fast geminate and slower second-order rebinding phases. Two slow phases were observed, having time constants consistent with reported binding rates for R and T state molecules. The geminate yield was 50%, the majority (37%) rebinding with a 70 ns time constant, consistent with previous studies, but an additional low-amplitude (13%) phase was resolved, with an 890 ns time-constant. Difference FTIR bands are also observed in the 1300-1700 cm -1 region, where protein vibrations are expected. In the nanosecond regime these bands varied irregularly, due to instrument limitations, but in the microsecond regime they evolved (30 µs time constant) toward the static difference spectrum of HbCO minus deoxyHb, reflecting the expected evolution from R to T state photoproduct molecules. The difference spectra of R and T photoproduct molecules extracted from the data via kinetic analysis contain not only common bands but also bands that are distinctive. The R photoproduct difference spectrum contains a positive/negative band pair at 1649 and 1683 cm -1 , which is interpreted as resulting from the breaking of one or more R-helical carbonyl H-bonds. Candidate H-bonds are those that connect the H-helix residues Tyr R140 and β145 with the F-helix residues Val R93 and β95, in both HbCO and deoxyHb. These H-bonds are believed to break and reform at intermediate stages of the allosteric pathway, on the basis of UV Raman evidence.

Time-resolved resonance Raman (TR 3 ) spectra are obtained with a pair of Q-switched Nd : YLF-pumped Ti : sapphire lasers, generating tunable (810-920 nm) ∼20 ns pulses at a 1 kHz repetition rate. Frequency doubling in lithium borate (LBO) provides blue (405-460 nm) pump and probe pulses, while UV probe pulses, tunable from 205 to 230 nm, can be generated by doubling the second harmonic in b-barium borate (BBO). Pump and probe pulse timing are controlled electronically. A timing sequence is implemented in which exposure of the multichannel detector alternates between positive and negative time delays between pump and probe pulses, so that accumulated difference spectra are free of artifacts from spectrograph drift or gradual decomposition of the sample. The system was tested on the carbonmonoxy hemoglobin (HbCO) photocycle, for which UV TR 3 spectra have previously been reported. HbCO was pumped at 419 nm, at the maxima of the strong Soret absorption band, and saturation of the photoresponse (maximum deligation) was established by measuring the intensity ratio of the HbCO and deoxyHb n 4 porphyrin RR bands, generated with 425 nm probe pulses. UV TR 3 difference spectra were obtained at time intervals from 0.06 to 20 µs using 229 nm probe pulses. They are in good agreement with those recorded previously with a pair of 300 Hz excimer-dye lasers. The time required to achieve a comparable signal-to-noise ratio was eight times shorter with the 1 kHz Nd : YLF-Ti : S lasers.