Microperoxidase-11 has been immobilized on siliceous materials MCM-41 and SBA-15 and on amino-functionalized SBA-15. Resonance Raman spectroscopy has provided solid evidence that the exogenous species occupy the pores of the mesoporous silica materials. Photoreduction of the microperoxidase-11 Fe-III center has been observed to occur in the immobilized samples and results in a long-lived stable reduced heme. Reoxidation of the heme occurs upon addition of oxygen, and the redox cycle can be repeated numerous times. The source of the electron resulting in reduction of the heme is proposed to originate from the silica matrix, and functionalization of silica surface is suggested to facilitate electron transfer to the heme.

FTIR difference spectroscopy is used to reveal changes in the internal structure and amino acid protonation states of bovine cytochrome c oxidase (CcO) that occur upon photolysis of the CO adduct of the two-electron reduced (mixed valence, MV) and four-electron reduced (fully reduced, FR) forms of the enzyme. FTIR difference spectra were obtained in D2O (pH 6–9.3) between the MV-CO adduct (heme a3 and CuB reduced; heme a and CuA oxidized) and a photostationary state in which the MV-CO enzyme is photodissociated under constant illumination. In the photostationary state, part of the enzyme population has heme a3 oxidized and heme a reduced. In MV-CO, the frequency of the stretch mode of CO bound to ferrous heme a3 decreases from 1965.3 cm−1 at pH* ≤7 to 1963.7 cm−1 at pH* 9.3. In the CO adduct of the fully reduced enzyme (FR-CO), the CO stretching frequency is observed at 1963.46±0.05 cm−1, independent of pH. This indicates that in MV-CO there is a group proximal to heme a that deprotonates with a pKa of about 8.3, but that remains protonated over the entire pH* range 6–9.3 in FR-CO. The pKa of this group is therefore strongly coupled to the redox state of heme a. Following photodissociation of CO from heme a3 in MV oxidases, the extent of electron transfer from heme a3 to heme a shows a pH-dependent phase between pH 7 and 9, and a pH-independent phase at all pH's. The FTIR difference spectrum resulting from photolysis of MV-CO exhibits vibrational features of the protein backbone and side chains associated with (1) the loss of CO by the a3 heme in the absence of electron transfer, (2) the pH-independent phase of the electron transfer, and (3) the pH-dependent phase of the electron transfer. Many infrared features change intensity or frequency during both electron transfer phases and thus appear as positive or negative features in the difference spectra. In particular, a negative band at 1735 cm−1 and a positive band at 1412 cm−1 are consistent with the deprotonation of the acidic residue E242. Positive features at 1552 and 1661 cm−1 are due to amide backbone modes. Other positive and negative features between 1600 and 1700 cm−1 are consistent with redox-induced shifts in heme formyl vibrations, and the redox-linked protonation of an arginine residue, accompanying electron transfer from heme a3 to heme a. An arginine could be the residue responsible for the pH-dependent shift in the carbonyl frequency of MV-CO. Specific possibilities as to the functional significance of these observations are discussed.

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu(B) binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu(B). Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu(B) and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 micros resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa(3) and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu(B)-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O(2) in flow-flash experiments.

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.

By coupling an infrared diode laser to a conventional infrared microscope, one achieves resolution approaching the diffraction limit while enabling rapid data collection. This technique is demonstrated with the use of a layered polymer sample that has been contaminated by migration of a volatile additive from an exogenous source. The distribution of this additive in the layered structure is shown to correlate with specific layers and reveals a concentration gradient suggesting a diffusive mechanism of additive migration parallel to the layered structure.

Difference FTIR spectroscopy was used to study protein changes associated with carbon monoxide (CO) photodissociation in two heme-copper oxidases, cytochrome c Oxidase (CcO) isolated from bovine heart muscle and Cytochrome bo (Cbo) isolated from Escherichia coli. Low temperature infrared data were obtained before and after photodissociation of CO from the heme iron in CcO. The resulting light-minus-dark difference spectra contain changes associated with the transfer of the CO ligand from iron to copper, and any accompanying protein and cofactor changes associated with this exogeneous ligand transfer. Time-resolved (TR) difference data were obtained at room temperature after photolysis of CO from the heme iron. This data reveals protein and heme changes occurring on the timescales of 5 us to 20 ms. TR difference FTIR data obtained on the two species of heme-copper oxidases is discussed.

Photodissociation of fully reduced, carbonmonoxy cytochrome bo3 causes ultrafast transfer of carbon monoxide (C triple bond O) from heme iron to CuB in the binuclear site. At low temperatures, the C triple bond O remains bound to CuB for extended times. Here, we show that the binding of C triple bond O to CuB perturbs the IR stretch of an un-ionized carboxylic acid residue, which is identified as Glu286 by mutation to Asp or to Cys. Before photodissociation, the carbonyl (C=O)-stretching frequency of this carboxylic acid residue is 1726 cm-1 for Glu286 and 1759 cm-1 for Glu286Asp. These frequencies are definitive evidence for un-ionized R-COOH and suggest that the carboxylic acids are hydrogen-bonded, though more extensively in Glu286. In Glu286Cys, this IR feature is lost altogether. We ascribe the frequency shifts in the C=O IR absorptions to the effects of binding photodissociated C triple bond O to CuB, which are relay ed to the 286 locus. Conversely, the 2065 cm-1 C triple bond O stretch of CuB-CO is markedly affected by both mutations. These effects are ascribed to changes in the Lewis acidity of CuB, or to displacement of a CuB histidine ligand by C triple bond O. C triple bond O binding to CuB also induces a downshift of an IR band which can be attributed to an aromatic C-H stretch, possibly of histidine imidazole, at about 3140 cm-1. The results suggest an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286. A chain of bound water molecules may provide such a connection, which is of interest in the context of the proton pump mechanism of the heme-copper oxidases.

Mononuclear M(III) complexes of formulae [MCp*Cl-(3-n)(R(2)pzH)(n)]((n-1)+) (M = Rh or Ir; n = 1,2) have been prepared through reactions of pyrazole (pzH, i.e. R = H) or substituted pyrazoles (R = Me or t-butyl) with rhodium and iridium cyclopentadienyl or pentamethylcyclopentadienyl precursors; for n = 1, the crystal and molecular structure has been determined by using X-ray diffraction (11, M = Rh, R=H, FW = 377.96g mol(-1) space group P2(1)/n, a = 7.1685(7), b = 15.740(3), c = 13.407(4)Angstrom, beta = 94.50(3)degrees), Dinuclear complexes of formulae [M(eta(5)-C(5)R(5))Cl(mu-pz)](2) (18, M = Rh, R = H; 19, M = Rh, R = H; 21, M = Ir, R = H; 20, M = Ir, R = Me; 21, M = Ir, R = H) containing pyrazolyl bridges can be isolated through further reaction of the mononuclear compounds, or more directly from the chloro-bridged dimers [M(eta(5)-C(5)R(5))Cl-2](2) by treatment with pyrazole in the presence of Et(3)N, although the dipyrazole iridium cation [IrCp*Cl(pzH)(2)](+) (16) does not undergo this type of dimerization. The dimeric complexes, which possess a 'chair' geometry about the 6-membered bridging heterometallocycle, have been shown to undergo a core conformational change ('chair':'boat') upon chemical reduction or halide abstraction. Chloride abstraction from 18 yields the binuclear product [{RhCp*(mu-pz)}(2)(mu-Cl)]BF4 (23) and reduction of either 18 or the C5H5 analog 19 gives access to the metal-metal bonded binuclear complexes [Rh(eta(5)-C(5)R(5))(mu-pz)](2), 25 (R = Me) and 26 (R = H), which adopt a 'boat' core conformation. The reactivity of the metal-metal bonded products has been investigated: a triply-bridged single-fragment oxidative addition product resulting from reaction with H+/MeOH ({[RhCp(mu-pz)](2)(mu-OMe)}O2CCF3, 29) has been structurally characterized (FW = 614.2 g mol(-1), space group P2(1)/n, a = 12.9647(3), b = 13.805(3), c = 11.593(3) Angstrom, beta = 90.44(2)degrees).

A high-pressure stopped-flow apparatus developed in our laboratories provides the capability to use dissolved gaseous reactants at elevated concentrations in solution (in equilibrium with gas pressures up to ca. 30 atm) for measurement of reaction kinetics. We have used this apparatus to follow the reaction of dioxygen with bovine cytochrome c oxidase following photolysis of the fully reduced CO ligated enzyme up to a dioxygen concentration of 16 mM. The observed rate dependence on [02] follows saturation kinetics and was fit to a limiting rate of 1.0 X 10(6) s(-1). This value is approximately the same as that for the thermal loss of CO to solution from the transient CuB bound state formed upon photolysis of the heme-CO complex. Implications for the mechanism of O2 binding and reduction by the heme-copper oxidases are discussed.