We have studied the infrared spectra of the bound and photodissociated states ofMb-"2CO and Mb-'3CO from 5.2 to 300 K. The absorbance peaks seen between 1800 and 2200 cm-1 correspond to CO stretching vibrations. In the bound state of Mb-'2CO, the known lines A0 at 1969, A1 at 1945, and A2 at 1927 cm-1, have center frequencies, widths, and absorbances that are independent of temperature between 5.2 and 160 K. Above 160 K, A2 gradually shifts to 1933 cm-'. The low-temperature photodissociated state (Mb*) shows three lines (BO, B1, B2) at 2144, 2131, and 2119 cm-' for "2CO. The absorbances of the three lines depend on temperature. Bo is tentatively assigned to free CO in the heme pocket and B1 and B2, to CO weakly bound to the heme or heme pocket wall. The data are consistent with a model in which photodissociation of MbCO leads to B1 and B2. B2 decays thermally to B1 above 13 K; rebinding to A occurs from B1. The barriers between B2 and B1 and between B1 and A are described by activation enthalpy spectra. Heme and the central metal atom in state Mb* have near-infrared, EPR, and Mossbauer spectra that differ slightly from those of deoxyMb. The observation of essentially free CO in state B implies that the difference between Mb* and deoxyMb is not due to an interaction of the flashed-off ligand with the protein but is caused by an incomplete relaxation of the protein structure at low temperatures.The reversible binding of CO to the storage protein Mb can be studied with flash photolysis (1). Experiments in which the Soret line was monitored demonstrate that the binding process involves a number ofsteps (2, 3). Here we show that monitoring the CO stretching vibration reveals additional features of the protein's interior.The active center of Mb, the heme group, is embedded in the protein (Fig. 1) (4) and the ligand binds at the central heme iron. In flash photolysis, the bound-state MbCO is photodissociated. Below 200 K the CO cannot leave the heme pocket and rebinds from there. Two states are involved in low-temperature recombination: state A, in which the CO is bound, the heme is nearly planar, and the iron atom has spin 0; and state B, in which the CO is photodissociated from the heme iron and remains in the protein pocket and the iron has spin 2. At low temperatures, the rebinding process B to A is not exponential in time. We have explained this observation by postulating the existence of conformational substates (2, 5). At low temperatures each Mb molecule is frozen into a particular substate with a specific barrier height for rebinding. From 180 to 80 K the transition occurs by an over-the-barrier Arrhenius process; below 60 K, quantum mechanical tunneling dominates (6, 7). State B (Mb*) has been studied in MbCO and CoMbCO by near-infrared (8, 9), EPR (10, 11), and Mbssbauer (12) Pentex (Kankakee, IL) was dissolved in 70% (vol/vol) glycerol in water buffered to pH 7 with 0.1 M phosphate. The sample was stirred under a CO atmosphere for several hours, reduced with sodium dithionite, and stirred for s...

Carbon monoxide bound to iron or copper in substrate-reduced mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) from beef heart has been used to explore the structural interaction of the a3 heme-copper pocket at 15 K and 80 K in the dark and in the presence of visible light. The vibrational absorptions of CO measured by a Fourier transform infrared interferometer occur in the dark at 1963 cm-1, with small absorptions near 1952 cm-1, and are due to a3 heme--CO complexes. These disappear in strong visible light and are replaced by a major absorption at 2062 cm-1 and a minor one at 2043 cm-1 due to Cu--CO. Relaxation in the dark is rapid and quantitative at 210 K, but becomes negligible below 140 K. The multiple absorptions indicate structural heterogeneity of cytochrome oxidase in mitochondria. The Cu--CO absorptions (vCO) are similar to those in hemocyanin--CO complexes from molluscs (vCO - 2062 cm-1) and crustaceans (vCO = 2043 cm-1). The 2062 cm-1 Cu--CO absorption of cytochrome oxidase is split into two bands at 15 K. Analysis of spectral data suggest the presence of a very nonpolar heme--Cu pocket in which the heme-CO complex is highly ordered, but in which the Cu--CO complex is much more flexible, especially above 80 K. A function for these structures in oxygen reduction is proposed.

The intramolecular binding of carbon monoxide ( 13 C 16 0/ 12 C 16 0 and 12 C 18 0/ 12 C le O mixtures) to myoglobin at 20 and 60 K is observed with time-resolved Fourier-transform infrared spectroscopy. The binding rates (at » 1 ks) for the molecules 12 C le O, 12 C 18 O f and 13 C 16 0 are in the ratio 1:0.84:0.65 at 20 K, 1:0.87:0.84 at 60 K. The large isotope effect confirms earlier evidence for molecular tunneling. The fact that 12 C ls O binds faster than 13 C 16 0 implies that structure effects are significant in molecular tunneling.

The SH vibrational absorption of cysteine F9(beta-93) in concentrated aqueous solutions of native liganded hemoglobin (human HbA, horse, and bovine) has been observed by use of Fourier transform infrared spectroscopy. The pattern of beta-93 SH absorption intensity is ligand dependent. In bovine hemoglobin derivatives the SH absorption intensity pattern is (carbonmonoxy)hemoglobin (HbCO) greater than oxyhemoglobin (HbO2) = cyanomethemoglobin (HbCN) much greater than aquomethemoglobin (metHb) and deoxyhemoglobin (deoxyHb). In horse and human hemoglobin derivatives the pattern is HbCO greater than or equal to HbO2 greater than HbCN greater than metHb. The bovine metHb beta-93 SH shows a much lower absorptivity than that of horse or human metHb, and thus it has a different local tertiary equilibrium conformation than does horse or human hemoglobin. X-ray diffraction studies have shown the beta-93 SH in carbon monoxide or oxygen bound hemoglobin to be situated within a nonpolar pocket between the F, G, and H helices. The higher than usual SH absorption frequency (2592 cm-1) that we observe implies there is no hydrogen bonding for this thiol group while situated within this nonpolar pocket. A similar beta-93 SH absorption has been observed in the beta-chain tetramer (thalassemic hemoglobin H in vivo). The beta-112 SH stretching band, previously observed in the alpha 2 beta 2 tetramer, was observed for the first time in the beta-chain tetramer. A band at 2610 cm-1 that is not due to SH was resolved and found to be ligand dependent.

Interstitial lung disease compatible with idiopathic pulmonary fibrosis (IPF) developed in 19 previously healthy patients. Although interstitial and/or honeycomb parenchymal fibrosis was present in all, there were patchy areas of paucicellular septal capillary injury along with corroborative direct immunofluorescent evidence of a humorally mediated microvascular injury syndrome. Significantly elevated factor VIII levels were seen in 17 of 18 patients tested. Antiphospholipids were present in all 18 patients tested, comprising antibodies of phosphatidylethanolamine, beta-2 glycoprotein, phosphatidylcholine, and/or phosphatidylserine. Anti-Ro and/or anti-ribonucleoprotein (RNP) antibodies were seen in 4 patients. Serologic evidence of infection with cytomegalovirus (CMV) was found in 9 patients and parvovirus B19 (B19) in 9 patients; 1 patient was not tested. Molecular studies revealed B19 DNA in 6 of 6 B19-seropositive patients. In situ hybridization studies revealed CMV RNA in pulmonary cells in patients with serologic evidence of active CMV infection despite the absence of cytopathic changes typical of CMV infection. Antiphospholipid antibodies, antiendothelial cell antibodies, and/or endotheliotropic viral infections related to B19 and CMV may be of pathogenetic importance to the evolution of IPF. This report underscores the potential importance of microvascular injury in the evolution of IPF.