Program and Abstracts of Joint Meeting Biophysical Society and American Physical Society, Washington, DC, March 27–30, 1979

1978 Biophysical Journal  
X-ray absorption spectra of metalloproteins have been made at the Stanford Synchrotron Radiation Laboratory. The Extended X-ray Absorption Fine Structure was measured and used to determine bond lengths. Methods of data analysis will be discussed briefly and in particular it will be shown how Fourier filtering combined with independent knowledge of the phase shifts allows determinations of distances to the first shell of neighbors. In carbonic anhydrase it will be shown that an iodide ion,
more » ... as an inhibitor, is bonded directly to the zinc atom, at a distance of 2.65±0.06X. In deoxyhemoglobin the Fe-N porphyrin distance was determined to be 2.055+0.01 while in oxyhemoglobin it was 1.98±0.01. Corrections which remove from the data contributions from axial ligands of +0.01 and -0.04O respectively are included. In deoxyhemoglobin these distances are consistent with the iron being up to 0.3X above the nitrogen plane, with the best value being 0.2X. These distances are identical with those measured simultaneously on the oxygenated and deoxygenated forms of "picket fence" porphyrins. In rubredoxin, containing one iron bound to four cysteinyl sulfurs, the average Fe-S distance is determine to be 2.26±0.01 in the oxidized state both in the powder and in solution. Upon reduction, in solution, it was 2.32±0.02L. In the oxidized state detailed analysis showed that all four Fe-S bonds were the same length to within the errors, which were ±0.10. In all three proteins these results represent refinements and modifications of the structures as previously determined by X-ray crystallography. Photosynthesis uses solar energy to split water to provide reduced substances and molecular oxygen. This process is poorly understood at the molecular level. In the water-splitting reaction it is known that metastable interm ediates are produced following light absorption; m anganese and chloride ion are i m plicated as essential catalytic cofactors. C oncerted efforts to observe m em brane-bound M n by ESR spectroscopy have failed. M anganese is, in this instance, one of the refractory, "spectroscopically silent" atoms. We have inaugurated X-ray spectroscopic studies of m anganese in chloroplast m em branes to seek inform ation on the oxidation state or states of M n, its ligands, and any changes thereof which accom pany the oscillatory production of 02 following a series of brief saturating light flashes. It has been know n for m any years that m olybdenu m is required for the growth of nitrogen fixing organism s. While molybdenum is invariably present in active preparations of purified nitrogenase, its function rem ains unclear because of its spectroscopic silence. Studies on nitrogenase model systems containing molybdenum provide circumstantial evidence to support the contention that it plays a central role in the reduction mechanism but no direct evidence has been presented which demonstrates the involvement of the metal in any nitrogenase reaction. We have examined the X-ray spectrum of molybdenum in nitrogenase isolated from Azotobacter vinelandii with the goals of determining if substrates, inhibitors, and cofactors interact directly with the meta], change its oxidation state(s), or both. X-ray absorption edge spectroscopy contains information about a) charge of the absorbing atom, b) degree of covalency of bonds, and c) coordination geometry. This technique has been used to investigate the various oxidation states of metal atoms involved in oxygen binding of the respiratory proteins cytochrome oxidase (two coppers and two irons) and hemocyanin (two coppers). The extended x-ray absorption fine structure technique (EXAFS) yields information about a) the average distance from the absorbing atom to each coordination shell, b) magnitude of neighbor atom distribution about the average distance, c) number of atoms in each coordination shell, and d) the Debye-Waller factor. Hemocyanin has been observed by this technique and model compounds for the proteins have been investigated. The models include polar and covalent compounds of Cu(I) and Cu(II); Fe(II) and Fe(III) complexes of tetraphenyl porphorin and protoporphorin IX dianion having 5th (and 6th) ligands polar and/or covalent; and Cu-Cu, Fe-Fe, and Fe-Cu complexes. Calcium coordination has been studied with model compounds and a correlation between edge energy, average bond distance, and coordination number found. Ca++-EDTA complex and CaC92 solution have been investigated and studies of phospholipid bilayers containing calcium have been made as functions of temperature and CaC22 solution content. Preliminary studies have also been conducted on both calcium activated and calcium modulated proteins. X-ray absorption spectroscopy is a technique which can examine the electronic and structural environment of a particular element in any physical state. The availability of a high intensity, broad band synchrotron radiation source at Stanford has made data collection on dilute metallo protein samples feasible for the first time. Analysis of the data provides accurate distances to neighboring atoms and coordination numbers as well as qualitative identification of the type of ligands. This paper will discuss the applications of this technique to the study of the Mo environment in the nitrogenase enzyme system and of the Cu environment in the proteins hemocyanin azuri n. Biomolecules provide a nearly ideal laboratory for investigations of Low-temperature reactions. Reactions, such as dissociation and rebinding of small ligands to heme proteins, can be initiated by light pulses and followed by monitoring optical absorbance changes. Below about 200 K, the processes after photodissociation are intramolecular. Rebinding is nonexponential in time, thus providing evidence for distributed barriers. The probability densities for the distributions can be determined and turn out to be characteristic for the protein structures. The presence of distributions is explained by postulating that biomolecules can exist in many different conformational states, with slightly different structures and functional properties. Studies as function of pressure and temperature give information about conformational relaxation. Rebinding can be observed down to 2 K; below about 30 K, and in some cases below about 100 K, it must occur by quantum-mechanical molecular tunneling. The distributed nature of the tunneling barrier allows a study of tunneling as function of barrier height. W-PM-2M ELECTRON TUNNELING WITH VIBRONIC COUPLING: THEORY, EXPERIMENTS, AND BIOLOGY. The theory of electron tunneling between two fixed sites requires vibronic coupling to provide energy conservation between initial and final states. The transfer rate is the product of a tunneling matrix element from electron wave function overlap and a vibronic factor, involving vibronic coupling parameters but independent of the tunneling matrix element. The theory is closely related to non-adiabatic outer sphere transfer of electrochemistry. Applied to charge separation in bacterial photosynthesis, the theory describes why some electron transfers have activation energies and others do not, how the quantum efficiency for charge separation is made high, and predicts approximate distance between the various sites. According to the theory, a weak transfer band, of molar extinction coefficient .1, should exist for biological electron transfer complexes. Such bands have been observed in model and in biological systems, and their observed properties are a quantitative test of the electron transfer theory. The primary process of vision is initiated with the absorption of a photon by the molecular photoreceptor, rhodopsin, resulting in the formation of a new intermediate, prelumirhodopsin. Picosecond kinetic studies of this photochemical event indicate that the process involves a proton translocation. At room temperature prelumirhodopsin (A max, 543 nm) is formed with 6 x 10-12 sec 259) is 8.3x103(M sec)l yith no significant dependence on either ionic strength or pH. The activation parameters are: AH =2.3 kcal. mole-1 and AS*=-33 eu. The self-exchange ET rate between Cocyt c and Co-cyt c+ was found to be less than 133 (M sec)-l from the relaxation time measurement of the pulsed pmr resonance of Met-80. The ET between Co-cyt c and Fe(EDTA)has a second order rate constant of 68.3 (M sec)-l (250, p=0.1, pH 7 phosphate) and AH*=4.2 kcal. mole1I and AS'--36 eu. From the ionic strength dependence an effective "active-site charge" of +0.45 was estimated for Co-cyt c . The autoxidation of Co-cyt c was found to have a rate constant at 250 of 12.3 (M sec)-l and independent of ionic strength. The reduction of methemoglobin by Co-cyt c was studied using nine mediators of different redox potentials. With phenazine methosulfate, the rate constant is 2.9x104 (M sec)1l (250, pH 7, O.1M phosphate) and AH*i7 kcal. mole-1 and AS*=-18 eu. These results are compared with the corresponding ET processes involving the native proteins and found to be in excellent agreement with theories.
doi:10.1016/s0006-3495(78)85531-3 fatcat:47utedebojfjphn7iqnmj4dlnq