M?ssbauer spectroscopy on nonequilibrium states of myoglobin: a study of r-t relaxation.

A frozen solution of 57Fe-enriched metmyoglobin was irradiated by x rays at 77 K. Mössbauer spectra showed a reduction of Fe(III) high spin by thermalized electrons and a production of a metastable Fe(II) low spin myoglobin complex with H2O at its sixth coordination site. The relaxation of the intermediate was investigated by Mössbauer spectroscopy as a function of temperature and time. The relaxation process starts above 140 K and is fully completed at approximately 200 K. At temperatures between 140 and 200 K, the relaxation lasts for hours and is nonexponential in time. Up to 180 K, the process can be described satisfactorily by a gamma distribution of activation enthalpies with an Arrhenius relation for the rate coefficient. The temperature and time dependence of the Mössbauer parameters indicates structural changes in the active center of the protein as early as 109 K that continue for several hours at higher temperatures. Above 180 K, structural rearrangements involving the whole protein molecule lead to a shift and narrowing of the barrier height distribution.     

Dynamic evaluation of the incidence of hereditary pathology by an accounting of spontaneous abortions and congenital developmental defects

A new method is suggested to control the mutation process in man by the dynamics of hereditary pathology frequency. The paper presents data on registration of spontaneous abortions, congenital malformations, Down's syndrome and perinatal death in Angarsk for 10 years (1971-1980). No changes are found in the frequencies of these data. The dynamic analysis of hereditary pathology "units" of mutation origin for 10 years did not show any time trend.     

Protein dynamics in an intermediate state of myoglobin: optical absorption, resonance Raman spectroscopy, and x-ray structure analysis

A metastable state of myoglobin is produced by reduction of metmyoglobin at low temperatures. This is done either by irradiation with x-rays at 80 K or by electron transfer from photoexcited tris(2, 2'-bipyridine)-ruthenium(II) at 20 K. At temperatures above 150 K, the conformational transition toward the equilibrium deoxymyoglobin is observed. X-ray crystallography, Raman spectroscopy, and temperature-dependent optical absorption spectroscopy show that the metastable state has a six-ligated iron low-spin center. The x-ray structure at 115K proves the similarity of the metastable state with metmyoglobin. The Raman spectra yield the high-frequency vibronic modes and give additional information about the distortion of the heme. Analysis of the temperature dependence of the line shape of the Soret band reveals that a relaxation within the metastable state starts at approximately 120 K. Parameters representative of static properties of the intermediate state are close to those of CO-ligated myoglobin, while parameters representative of dynamics are close to deoxymyoglobin. Thus within the metastable state the relaxation to the equilibrium is initiated by changes in the dynamic properties of the active site.     

Protein dynamics of a β-sheet protein

Rhodnius prolixus Nitrophorin 4 (abbreviated NP4) is an almost pure β-sheet heme protein. Its dynamics is investigated by X-ray structure determination at eight different temperatures from 122 to 304 K and by means of Mössbauer spectroscopy. A comparison of this β-sheet protein with the pure α-helical protein myoglobin (abbreviated Mbmet) is performed. The mean square displacement derived from the Mössbauer spectra increases linearly with temperature below a characteristic temperature T _c. It is about 10 K larger than that of myoglobin. Above T _c the mean square displacements increase dramatically. The Mössbauer spectra are analyzed by a two state model. The increased mean square displacements are caused by very slow motions occurring on a time scale faster than 140 ns. With respect to these motions NP4 shows the same protein specific modes as Mbmet. There is, however, a difference in the fast vibration regime. The B values found in the X-ray structures vary linearly over the entire temperature range. The mean square displacements in NP4 increase with slopes which are 60% larger than those observed for Mbmet. This indicates that nitrophorin has a larger structural distribution which makes it more flexible than myoglobin.     

A water network within a protein: temperature-dependent water ligation in H64V-metmyoglobin and relaxation to deoxymyoglobin

The sperm whale myoglobin mutant H64V, where the distal histidine is mutated to valine, is known to be five coordinated in the ferric state at room temperature and physiological pH. A change of the ligation in this H64V-Mbmet has been observed by optical absorption spectroscopy as a function of temperature from 20 K to 300 K. Above the dynamical transition at about 180 K one observes the temperature-dependent equilibrium between five- and six-ligated heme. Below the dynamical transition the equilibrium is frozen-in at about 50% of six-coordinate molecules. The water ligation of the iron occurs at temperatures where protein-specific motions are present, as monitored by M?ssbauer spectroscopy. The X-ray structures of H64V-Mbmet at 300 K and 110 K are reported with a resolution of 1.5 A and 1.3 A, respectively. The measurements at high resolutions are possible owing to crystallization in the space group P2(1), whereas all mutant myoglobins studies up to now have been carried out with crystals in the space group P6. The overall structure at both temperatures is very close to the native myoglobin. The binding of water at the sixth coordination site at lower temperatures is possible owing to a stabilizing water network extending from the protein surface to the active centre. The reduction of the H64V-Mbmet by electrons obtained by X-ray irradiation of the water-glycerol solvent at 85 K produces an intermediate low-spin state of the water-ligated molecules where Fe(II) retains the six-fold coordination. M?ssbauer spectroscopy shows that the relaxation of the metastable low-spin state to high-spin H64V-Mbdeoxy with dissociation of the Fe(II)-H(2)O bond starts at about 115 K and is completed at about 170 K. Differences in the dynamics properties of the native and mutant myoglobin and the connection to the dynamical transition around 180 K are discussed.     

From metmyoglobin to deoxy myoglobin: relaxations of an intermediate state

Metmyoglobin has been reduced at low temperature (below 100 K) using x-rays or by excitation of tris(2,2′,bipyridine)ruthenium(II) chloride with visible light. Upon reduction, an intermediate state is formed where the structure of the protein is very similar to that of metmyoglobin with the water molecule still bound to the heme iron, but the iron is II low spin. The nature of the intermediate state has been investigated with optical spectroscopy. The Q_o and Q_v bands of the intermediate state are split, suggesting that the protoporphyrin is distorted. The intermediate state undergoes a relaxation observed by a shifting of the Soret band at temperatures above 80 K. Above 140 K, the protein begins to relax to the deoxy conformation. The relaxation kinetics of the protein have been monitored optically as a function of time and temperature from minutes to several hours and from 150 K to 190 K. By measuring the entire visible spectrum, we are able to distinguish between electron transfer processes and the protein relaxation from the intermediate state to deoxy myoglobin. The relaxation has been measured in both horse myoglobin and sperm whale myoglobin with the relaxation occurring on faster time scales in horse myoglobin. Both the reduction kinetics and the relaxation show non-exponential behavior. The reduction kinetics can be fit well to a stretched exponential. The structural relaxation from the intermediate state to the deoxy conformation shows a more complex, dynamical behavior and the reaction is most likely affected by the relaxation of the protein within the intermediate state. Received: 30 June 1997 / Accepted: 6 November 1997     

Study of the relaxation of nonequilibrium hemoglobin states by M?ssbauer spectroscopy

Nonequilibrium hemoglobin states formed at low-temperature (T = 77K) reduction of its derivatives (MetHb and HbO2) by thermolysed electrons have been studied by M?ssbauer spectroscopy. Relaxation of nonequilibrium states at the samples heating was observed. Correlation between the relaxation temperatures and the changes of protein dynamic structure determined from M?ssbauer data were stated.