Apparent oxidation-reduction potentials at pH 7.0 and 25 degrees C were determined using the H2-hydrogenase system with ferredoxins from the following sources: Clostridium pasteurianum, -403 mV; C tartarovorum, -424 mV; C. acidi-urici, -434 mV; Peptococcus aerogenes, -427 mV; Chromatium D, -482 mV (pH 8.0); B. polymyxa, Fd I, -377 mV, and Fd II, -422 mV; and spinach, -428 mV. The pH dependence of these values was variable, ranging from -2 to -24 mV/pH unit increase for different ferredoxins. Over the range of buffer concentrations between 0.05 and 0.2 M, the potentials did not vary significantly. The number of electrons transferred during reduction (as determined by integrations of EPR spectra and by dithionite titration) is 2 for the first five proteins, while potentiometric data for all the cases fit a Nernst equation for which n = 1. The E degrees' value for the redox indicator methylviologen at pH 7.4 was found to be -460 mV, according to both the H2-hydrogenase system and cyclic voltammetry, significantly different from the value previously reported at higher pH's. Additionally, the presence of C. pasteuranum ferredoxin appears to shift the E degrees value of methylviologen to even more negative values. An analysis of sources of error inherent with potential determinations with H2 and hydrogenase is presented. The electronic and EPR spectra of P. aerogenes ferredoxin, for which the x-ray structure has been published, are given here. It appears that the determination of potentials of ferredoxin and other low-potential porteins with the H2-hydrogenase system affords certain experimental advantages over alternative methods currently employed with these and similar substances.

The high intensity x-ray flux from the synchrotron radiation at the Stanford Synchrotron Radiation Project has been used to study the extended x-ray absorption fine structure (EXAFS) of the iron-sulfur protein Peptococcus aerogenes rubredoxin. Absorption measurements were made from 7080 eV, which is below the K-edge of iron, to about 650 eV above the edge and structure was obtained over the entire region. By means of a model iron-sulfur compound for evaluating the phase shifts, the variation of the absorption above the edge of lyophilized, oxidized rubredoxin was converted to iron-sulfur distances. The data were fitted with a least squares program to a model in which three distances R3 were kept equal and the fourth'R1 was allowed to differ. The mean square error was constant over a region of this parameter space, becoming twice as large at R3 = 2.217, RI = 2.389 and R3 = 2.268, RI = 2.108 A. These values, which are the extreme differences allowed by the present data, are definitely closer to being equal than those found by the determination of the x-ray diffraction crystal structure of the similar protein from Clostridium pasteurianum. However, the average distance from our experiment is in excellent agreement with the average distance from the crystal structure determination. Preliminary EXAFS measurements were also made on the oxidized rubredoxin in solution at pH 7.0. The spectra were unchanged, indicating that the average iron-sulfur distance change is <0.02 A. Upon reduction the average iron-sulfur bond length increased by about 0.05 A.Since the EXAFS measurements can give accurate determinations of distances in proteins both in crystals and solution, the technique should be widely applicable. Recently high fluxes of x-rays have become available in the form of synchrotron radiation from electron storage rings; in particular the work reported here was performed on the Stanford Storage Ring SPEAR. At SPEAR one has 104 to 105 times more intensity of tuneable x-rays than that available from standard x-ray tubes. This increase in intensity has allowed x-ray absorption to be successfully observed in dilute materials; specifically proteins and metalloporphyrins have been measured (1). In the present study we report the first determination of interatomic distances in a protein, namely, the iron-sulfur bond distances in rubredoxin from Peptococcus aerogenes.While x-ray absorption measurements have been made sporadically since 1931 (2), the measurements, their interpretation and their applications to dilute systems have only recently been extensively developed due to the work of Sayers, Stern, and Lytle (3) and the use of synchrotron sources (4). The experimental technique used in these studies is the same as previously reported (5) and essentially consists of a slit-channel-cut silicon monochromator system which transforms the incident white radiation into a tuneable monochromatic beam of 1 eV bandwidth. The energy of the output beam is determined through the Bragg relationship by the angle that the highly ...

Magnetic susceptibility and proton magnetic resonance spectra are reported for the oxidized and reduced forms of the iron-sulfur protein Bacillus polymyxa ferredoxin I. The magnetic susceptibility of the oxidized form indicates antiferromagnetic exchange coupling between component iron atoms that is quantitatively similar to that observed for the clostridial ferredoxins and for the [(C2Hs)4N2[Fe4S4(SCH2C6H5)41 analog. Contact-shifted resonances observed in the proton-magnetic-resonance spectra of oxidized and reduced forms of the B. polymyxa protein can-be correlated with the contact-shifted resonances of corresponding redox forms of the clostridial ferredoxins. Characteristics of the contact-shifted resonances observed in partially reduced B. polynmyxa ferredoxin I are compatible with a "slow" rate of electron exchange between redox forms, which suggests that the "fast" electron exchange earlier observed in the eight-iron clostridial ferredoxins may derive from an intramolecular component.In recent years, a new subdivision of bacterial ferredoxins containing only a single cluster of four iron and four labile sulfur atoms per molecule has become known; two members of this new subclass of bacterial ferredoxins have been isolated(1) from the facultative nitrogen fixing bacterium Bacilus polymyxa and their chemical, physical, and electron paramagnetic resonance (EPR) properties defined (1-4). Both of these proteins have molecular weights of about 9000 daltons and contain four iron atoms and four labile sulfur atoms per molecule. Both optical absorption and EPR spectra strongly suggest that the iron and labile sulfur atoms in the four-iron ferredoxin are arranged into a single tetrameric cluster similar to the two tetrameric clusters found in the more complex eightiron bacterial ferredoxins (5). The purpose of this study was to investigate the magnetic susceptibility and proton magnetic resonance (PMR) properties of one of these four-iron ferredoxins, B. polymyxa ferredoxin I, in order to further characterize this new type of iron-sulfur protein. It same basic geometrical type, a hypothesis that must necessarily be tested by crystallographic means and which emphasizes the role of the protein moiety in dictating the electron-transfer properties (potentials and specificities) of these proteins. MATERIALS AND METHODSThe B. polymyxa ferredoxin I employed in these studies was isolated and purified by methods previously described (1). Reductions were carried out using British Drug House Na2S204. Nuclear magnetic resonance (NMR) spectra for the most part were obtained on a Varian 220 MHz PMR spectrometer; searches to extreme low-field were carried out on a Bruker 90 MHz spectrometer. Magnetic susceptibilities were determined by previously described NMR techniques (6).

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