Frank G. Fiamingo

Photochemical dissociation in optically dense solutions: applications to photolyzed carboxymyoglobin (Mb*CO)

Photodissociation of ligands has made important contributions to the understanding of function and structure of heme proteins. Here we present a theory for photochemical dissociation that is not limited by the assumption of previous analyses of optically thin samples, and apply it to interpretation of the photodissociated state of carboxymyoglobin (Mb*CO). Equations are derived and presented in terms of the effects of absorbance, [log10(I0/I) = A, the probability of absorption of light quanta per unit surface area], and the potential for dissociation, D (maximum probability of photodissociation per unit surface area; a linear function in time of photolysis), for both monochromatic and polychromatic light sources. When monochromatic light is used, we show that for large absorbances (A > 2) the fractional photolysis increases as (log D)/A, and may appear to “saturate” even though well below completion. For polychromatic light intensities and absorbances, the theory predicts that the near-infrared tail of the absorbance band of carboxymyoglobin should be sufficiently transparent to allow the radiation to penetrate the sample, yet still have a significant absorptivity such that complete photodissociation is possible. An optically thick myoglobin-CO sample illuminated with a tungsten lamp was observed to behave somewhere between these two theories. These theoretical relations may be useful in the analysis of photolysis data from optically dense solutions and as a guide for future experimental design.

X-ray absorption spectroscopy of myoglobin and iron prophyrin derivatives

Diverse interpretations of the structure of carboxymyglobin and its photodissociation products have led us to a reexamination of problems of data collection and analysis of iron porphyrins and heme proteins by x-ray absorption spectroscopy. We have been especially concerned with the assignment of statistical weight to individual wave functions that contribute to the EXAFS spectral region. In the case of iron porphyrin derivatives, a single axial first shell atom contributes only a small fraction of the total wave form, and its definition may be fraught with uncertainties. We have therefore attempted to calculate least squares best fits of the sum of all significant wave form contributions to the total EXAFS spectrum of a series of iron porphyrin model compounds and myoglobin derivatives. This has required inclusion of wave forms for all porphyrin ring atoms in addition to substituent groups directly bonded to the porphyrin ring. Axial ligands have been more difficult to establish, except in the case of tightly bonded atoms, and those whose waveforms are enhanced by multiple scattering. Radial distances estimated for pyrrole nitrogens, alphacarbons, and meso-carbons, are all well described by single scattering theory, and correspond closely to crystallographically estimated distances. Apparent radial distance for beta-carbons, and gamma-carbons, are shorter than crystallographic distances by about 0.2 and 0.6 A, respectively. This is expected from multiple scattering phase shifts. The radial distances estimated for pyrrole nitrogens are sensitive to spin state of the iron, as predicted from crystallographic studies. This is clearly observed both with model compounds (derivatives of tetraphenyl porphyrin and octaethylporphyrin) as well as with myoglobin derivatives.

Instrumental barriers in biological Fourier transform infrared spectroscopy

Biological applications of infrared spectroscopy have pressed for ever greater instrumental capabilities in terms of spectral sensitivity and quantitative exactness. Improved instrumentation has provided measurement of many vibrational modes in biological samples that previously were lost in noise. With highly optimized sampling conditions, useful measurements have been made with a peak-to-peak noise level less than 5 microabsorbance (5×10−6 absorbance), at 0.5 cm−1 resolution. However, optical and instrumental instabilities often result in sine waves that are not totally removed by the ratio of sample to reference. These often limit effective spectral sensitivity to 50 or 100 microabsorbance, peak-to-peak, and constitute a non-random noise. Non-atmospheric absorptions, especially one at 1959 cm−1 with 0.8 cm−1 band width (FWHM) are reported. The latter is due to a trace impurity in the KBr beam splitter substrate and compensator plate. Improvements in instrumentation and sampling conditions are expected to yield measurements of absorption bands as small as 50 microabsorbance with excellent signal/noise.

Multiple Forms of Cytochrome C Oxidase Observed in Heart Tissue, Myocytes, and Mitochondria by Fourier Transform Infrared Spectroscopy

Cytochrome c oxidase is the terminal enzyme in electron transport. It catalyzes the transfer of four electrons from cytochrome c and four protons of unknown origin to dioxygen, which is converted into two molecules of water. (For a thorough review see Wikstrom et al., 1981). Mammalian cytochrome oxidase is a large Y-shaped molecule that spans the inner mitochondrial membrane (Deatherage et al., 1983; Kim et al., 1985), and consists of one copy of each of 1–13 subunits (Kadenbach et al., 1986; Kuhn-Nentwig and Kadenbach, 1985 & 1986; Buse et al., 1985; Capaldi and Zhang, 1986). The enzyme has two functional domains: cytochrome a, which contains one heme (aFe) and one copper (CuA); and cytochrome a3, which contains the remaining herae (a3Fe) and copper (CuB). Cytochrome a3 is the site of ligand binding and dioxygen reduction.

Low Temperature Photolysis And Recombination Kinetics Observed With Biological Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectroscopy of biological macromolecules provides structural information about single vibrating groups and their interactions with nearest neighbors.