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Spectroelectrochemical investigation of Azotobacter vinelandii bacterial ferritin
Bioelectrochemistry and Bioenergetics 44 Ž1998. 301–307
Short communication
Spectroelectrochemical investigation of Azotobacter Õinelandii bacterial ferritin He-Qing Huang
a,)
, Feng-Zhang Zhang a , Liang-Shu Xu a , Qing-Mei Lin b, Jing-Wei Huang c , Ding Zeng a b
a Department of Biology, Xiamen UniÕersity, Xiamen 361005, China Research Center of EnÕironment Science, Xiamen UniÕersity, Xiamen 361005, China c Department of Chemistry, Xiamen UniÕersity, Xiamen 361005, China
Received 3 October 1997; received in revised form 28 October 1997
Abstract Bacterial ferritin of Azotobacter Õinelandii ŽAvBF. contains a function in accepting electrons from platinum electrode directly for complete iron release in the absence of a mediator. The reduction potentials of electron tunnels of y125, y310, and y370 mV for iron release are determined by direct spectroelectrochemical technique, which suggests which should be defined as midpoint potentials of electron–tunnel–heme on the surface of protein shell. A kinetic study for complete iron release by the electrode reduction at y600 mV fits the zero-order reaction law. q 1998 Elsevier Science S.A. Keywords: Azotobacter Õinelandii; Bacterial ferritin; Reduction potential; Electron–tunnel–heme and kinetics of iron release; Spectra and mediatorless
1. Introduction Ferritin is found mostly in animal, plant and bacteria and has clearly been recognized by their physiological functions for iron metabolism and storage w1–4x, amino acid sequence similarity w5–7x, and characteristics of physical chemistry w5,8x. The molecular structure of ferritin consists of protein shell with a highly symmetrical multi˚ in overall diameter w2,5x and of an iron subunits of 120 A core containing a few thousand molecules of hydrated iron phosphate compound w2,9x. In vitro, the kinetics of iron release from ferritin has been studied by the stopped-flow technique w10,11x and the normal spectrophotometry w12– 14x in detail. A mechanism by diffusion manner and channel transport that has been widely accepted by most researchers on ferritin is that the reducers such as sodium dithionite w10,12x or reduced methyl viologen w11x and the chelating agents such as a X – a dipyridyl w10–13x or 1,10phenanthroline w15x must pass through the channel of the shell into the core so that the Fe 3q component of ferritin can be reduced within the core and released out of the shell w10,14x. )
Corresponding author.
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 7 . 0 0 0 9 8 - 6
Horse spleen ferritin ŽHSF. appeared reductive currentless by pulse polarography, and its mineral cores isolated from protein shell showed electrode activity by voltammetry w16x, meaning the shell is sluggish to a physical electrode. On the contrary, AvBF and HSF containing Fe 2q within the core can be directly oxidized by large protein oxidants such as cytochrome c and CuŽII. protein with passing through the channel of ferritin scarcely without any mediator aid, suggesting that there are electron tunnels on the protein shell for transferring electron w17x. Comparing the characteristics of physical chemistry among bacterial ferritin ŽBF., HSF, and plant ferritin w1–4x, BF is known to contain heme Ž6–12 hemerBF. w17,18x showing a , b and soret bands at 557.5, 527, and 425 nm from Azotobacter Õinelandii w5x, at 557, 526, and 417 nm from Rhodobacter capsulatus 37b6 w18x, and at 560, 530, and 417 nm from Escherichia coli K 12, W 2244 w19x and to have higher ratio Ž1:1.2–1.4. structure of phosphate to iron within the core than that Ž1:8–9. of HSF w15,16,20,21x. In addition, oxidized AvBF ŽAvBFo . is a hydrogenase with H 2-uptake activity of 450 H 2rAvBF w21x. The maximum absorbance peaks with an 8-nm shift to red between holoŽ417 nm. and reduced apo-ferritin Ž425 nm. or reduced holo-ferritin Ž425–426 nm. are found w13,19,21x, and their
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spectral properties are tightly related to different procedures of extraction and purification w13x, to variant pH medium w22x, and to Fe 2q numbers within the core w20,22x. Recently, the reduction potentials of y475 mV for heme reduction and of y420 mV for iron core reduction from AvBFo were determined by microcoulometry mediator w23x. A rate of the core potential shifting to negative as a form of y205 mV q Žy115 mVrpH. from HSF is obtained by Watt et al. w23x and Huang et al. w24x. But, the actual role in iron metabolism and storage by the heme, the redox potential, the heme and the electron tunnel still remains obscure. Here, we present evidences revealing that AvBFo contains three midpoint potentials of electron–tunnel–heme ŽETH. having capacities to accept directly electrons from platinum electrode for iron metabolism in the absence of a mediator. The kinetics for complete iron release by the electrode reduction at y600 mV fits the zero-order reaction law. 2. Experimental 2.1. Chemicals and materials The strain OP of A. Õinelandii was a gift from The Center for the Study of Nitrogen Fixation, College of Agricultural and Life Science, University of Wisconsin, Madison, USA. Sodium dithionite and a X – a dipyridyl etc. were from Sigma. Most chemicals for experiments are of analytical or spectral purity. 2.2. Cell culture and AÕBF purification A. Õinelandii was grown in a 100-l fermentor in modified burk medium. The cells were harvested by centrifugation in mid-log phase Ž18 h.. Raw AvBFo was first isolated by the methods of Bulen et al. w4x and Li et al. w25x, and further purified by Burgess et al. w26x as previously described for purifying nitrogenase from A. Õinelandii. The purified AvBF was known to exhibit a single band on the gel electrophoresis ŽPAGE. and show a single peak on the high- performance liquid chromatography ŽHPLC.. 2.3. Protein and metallic element analysis Protein concentration measurement is normally described by the Lowry method. Bovine serum albumin containing 99% purity is used for protein standard. The total number of Fe 3q within the shell is determined by an atomic absorbance spectrophotometer after the ferritin is nitrified, and the Fe 2q numbers within the core is measured by spectrophotometry at 520 nm as previously described by Stiefel and Watt w5x and Watt et al. w23x. 2.4. Apparatus and reduction of AÕBFo An anaerobic electrochemical cell consists of three electrodes and an anaerobic reaction vessel Žsee Fig. 1.. Ž1.
Fig. 1. An anaerobic spectroelectrochemical cell. ŽA. Cell; ŽB. Cuvette; ŽC. Outlet; ŽD. Saturated calomel electrode; ŽE. Isolated platinum counter electrode; ŽF. Working platinum electrode; ŽG. Magnetic stirred bar; ŽH. Magnetic stirrer; ŽI. Inlet; ŽJ. Rubber serum stopper for automatically evacuating and flushing with 99.9% Ar gas. A distance about 0.3–0.4 cm among the electrodes is maintained before use.
Electrodes w1x: a radiometer fiber-tipped saturated ŽKCl. calomel electrode ŽSCE. is used as the reference electrode for the electrochemical synthesis detector ŽESD. w2x. An isolated platinum counter electrode Žanode., w3x a working electrode Žcathode, 20 = 60 mm. of round platinum for supporting high reduction current, and w4x the distance of 0.4–0.5 cm among the electrodes is maintained for obtaining stable reduction current. Ž2. Cell: an anaerobic reaction vessel equipped with water thermostat is designed as a closed reaction cell, which is automatically evacuated and flushed with 99.9% Ar gas using an automatic instrument. UV–visible 240 recording spectrophotometer equipped with an anaerobic cuvette of thermostat filled by the Ar gas is used for recording spectra of AvBF at a controlled potential. The equilibrium position at each experimental point for iron reduction is obtained by the stable residual current, which is monitored by a high differential voltmeter, with the controlled potential for 5–10 min. All reaction system contains 0.01 M Tris–HCl in 0.05 M NaCl, pH 7.0 and mediatorless. All reaction temperature Ž308C. is always maintained by a water thermostat. The mixed solution for iron reduction is stirred by a magnetic stirrer at a constant rate Ž300 rpm.. 2.5. Kinetic studies of complete iron release Three milliliters AvBFo and excess a X – a dipyridyl are mixed by an anaerobic instrument and placed into the cell quickly by perfused gas tight syringe. The controlled potential at y600 mV for iron reduction and release is supplied by ESD. The mixed sample is placed into the anaerobic cuvette filled with 99.9% Ar for total Fe 2q numbers measured by spectrophotometry at 520 nm according to the given reaction time. All controlled potentials are converted into standard hydrogen potential before use. The reaction end is indicated by the maximum number of
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iron release per molecular AvBFo Ž1800 Fe 3qrAvBFo .. According to total iron numbers per molecular AvBFo , a 1:1 ratio of added extra phosphate to iron within the core is prepared to study the effect of phosphate on the rate of complete iron release. 2.6. Midpoint potential measurements of the electron tunnel Four milliliters AvBFo is separated on a G-25 Sephadex column Ž2 = 10 cm. previously equilibrated with 0.025 M Tris–HClr0.15 M NaCl, pH 7.0 for removing free iron and phosphate before use. The AvBFo and excess a X – a dipyridyl are mixed by the instrument for 10 min, then the mixture is placed into the cell quickly under anaerobic condition, and is directly reduced by the controlled potential from ESD. Equilibrium position at each experimental point is monitored by a voltmeter. The reaction end at each curve is completed by the maximum number of Fe 3q release per molecular AvBFo Ž1800 Fe 3qrAvBFo .. The midpoint potential measurement of electron tunnel is calculated from half of maximum numbers of iron release per molecular AvBFo Ž1800 Fe 3qrAvBFo . against the given controlled potential at each new experiment.
Fig. 2. Redox spectra of AvBFo ranging from 380 to 580 nm. Curve A: reduction spectrum of AvBFo with electrode reduction at y600 mV for 2 h. Curve B: reduction spectrum of AvBFo with excess dithionite reduction for 2 h. Curve C: spectrum of AvBFo . Curve D: spectrum of AvBFh o after AvBFrd is at least oxidized by air for 1 h. The AvBFrd is anaerobically separated on the G-25 column for removing unstable Fe 2q and phosphate within the core before being oxidized.
2.7. Spectrum measurement of half-oxidized AÕBF Two milliliters reduced AvBF ŽAvBFr . is oxidized by the air or the mixture gas of 21% O 2 and 79% Ar until the decreasing rate of absorbance keeps constant monitored by a spectrophotometer at 557 nm in 2 min, then, the sample called half-oxidized AvBF ŽAvBFho . is placed into the anaerobic cuvette for spectral measurement ranging from 380 to 580 nm. 2.8. Reduction rate measurement of AÕBFo An 8-ml sample of AvBFo is divided into two Ž4.0 ml each vessel. by a micropipette, then the first sample is placed into an anaerobic cell for reduction with controlled potential at y600 mV. The second one is placed into an anaerobic vessel containing excess dithionite for reduction. The reaction of both samples is carried out at a constant stirred rate Ž300 rpm. and 308C simultaneously, and their absorbances are measured by a spectrophotometer-equipped double light at 557 nm against the indicated time.
3. Results 3.1. Spectra and characteristics of AÕBF Fig. 2C shows clearly that AvBFo exhibits the characteristic absorbance peaks at 557 Ž a ., 526 Ž b ., and 417 ŽSoret band. nm. Fig. 2B,C shows that AvBFr absorbance with dithionite reduction ŽAvBFrd . at spectral range from
380 to 580 nm is higher than that of AvBFo , but, a shift of maximum absorbance peaks to red can be unlikely observed with reducing degree increment due to the complete Fe 2q component still retained within the core. Fig. 2A,B shows that AvBFr by the electrode reduction ŽAvBFre . at y600 mV appears similar the absorbance intensity and curve characteristics to that of AvBFrd . It is well seen that a similar reduction degree and rate of both AvBFrd and AvBFre can be easily obtained and controlled by the chemical reducers and the physical electrode. The plotted results in Fig. 3E,S show the reduction rate of AvBFo as a function of the reaction time monitored at 557 nm. Both projection curves in Fig. 3 indicate that two nearly equal reduction rates for the whole protein shell and the iron core in part by dithionite and the electrode reduction are observed within 40 min, meaning a rate of supplying electrons to the core by dithionite is equal to that the ferritin picks up from the electrode. Fig. 2D shows that the whole absorbance intensity of half-oxidized AvBF ŽAvBFho . at spectral range from 380 to 580 nm is lower than that of AvBFrd or AvBFre ŽFig. 2A,B. and is higher than that of AvBFo ŽFig. 2C., clearly suggesting that AvBFho cannot return reversibly to its original conformation even though the oxidization reaction lasts up to 1 h under the air, because the phosphate and iron component in the part within the core have been lost while the protein is separated in the column before being oxidized. From the curve results analysis in Figs. 1 and 2, we can draw a few interesting conclusions. Ž1. A reduced
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Fig. 3. Reduction rate measurements in the AvBFo monitored at 557 nm. Curve E: electrode reduction at y600 mV for 50 min. Curve S: dithionite reduction for 50 min.
rate of AvBFo is faster than a oxidized that of AvBFrd or AvBFre . Ž2. The oxidization process of AvBFrd and AvBFre exhibits a complex pathway Žsee Fig. 1A–D., which is known to appear at a fast rate for oxidization in initial stage within 40 min, but which cannot reflect a complete process and characteristics of ferritin oxidization because of a series of changes involving the rate of iron release, conformation, and self-regulation of protein shell with the number of iron release increment within the core w21x. Ž3. The AvBFho Žsee Fig. 2D. may be a transitional state between AvBFrd or AvBFre and AvBFo . 3.2. Midpoint potential measurement of electron tunnel The reduced mediator such as reduced methyl viologen or natural red by the concentration diffusion can rapidly reduce the various metallic clusters within the protein w27–29x, but not the physical electrode controlled by the ESD, because most metallic proteins are sluggish to directly pick up electrons from the normal physical electrode w30x. One of the biological functions of the threefold channels from the ferritin is to play an important role in the thoroughfare for exchange of small complex, which has been cited by iron deposition and release between apoand holo-ferritins w2,15,31x, but neither an iron chain nor an iron–phosphate has been reported to be located on the surface of the channel for picking up the electrons from the chemical reducers or the electrode. On the contrary, Fig. 4 shows that the Fe 3q component within the core of AvBFo is directly reduced into Fe 2q composition by the platinum electrode and released out of the shell by a X – a dipyridyl, meaning that there are electron tunnels on the surface of protein shell for picking up the electron from the electrode, and transferring them into the core for iron reduction. A whole iron core with reduction potentials at y420 mV from AvBF w32x and at y215 mV from HSF w24x can be fully reduced by various reducers such as ascorbic acid Žy12 mV., FADH 2 Žy210 mV., FMNH 2 Žy250 mV., reduced methyl viologen
Žy416 mV., and dithionite Žy512 mV. w30,33x. In addition, a factor about tenfold for the rate of iron release is accelerated by a mixed reducers that both reduced methyl viologen and dithionite w11x. As these results above mentioned, an interesting conclusion is drawn that the AvBFo may exist in multi-reduction potentials of the channel and of the electron tunnels on the surface of the shell for iron reduction. The results in Fig. 4A show that the Fe 3q component within the core begin to be reduced into Fe 2q composition while the potential giving q100 mV VS P NHE is obtained by ESD, but the actual, about 5% of original Fe 3q component, is only reduced, and most Fe 3q–phosphate complex with the core is unlikely reduced at the indicated potential. Two additional experimental results have shown that the complex of Fe 2q Žbipy. 3 that have been released out of the protein shell appears reductive currentless by the given potential, and the free complex of Fe 2q ions and phosphate can be unlikely found in the medium when the ferritin is reduced, because none of the reduction current that increases drastically is obtained in the initial stage of iron reduction. In addition, regular kinetic properties for complete iron release have been cited w13x, which these results have clearly indicated, that the reductive current monitored by ESD come from the Fe 3q component reduction within the core, and that a stable remnant current, ranging from 20–100 m A, as well as a constant absorbance monitored at 520 nm by the spectrophotometer within 5 min, shows an equilibrium system of iron reduction for the core reduction at the given potential, and that a more negative potential needs to be obtained by ESD so that the remnant iron within the core can be further reduced as the potential of AvBFo shifts to negative with numbers of released iron within the core increment. AvBFo not only contains the three channels for iron release and
Fig. 4. The midpoint potential measurements of electron tunnels from AvBFo by electrode reduction. The three midpoint potentials of electron tunnel of y115 ŽA., y310 ŽB., and y370 ŽC. mV on the surface of the protein shell are determined by direct spectroelectrochemical technique in the absence of mediator and of other chemical, as well as biological reducers, respectively.
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deposition, but also have the three electron tunnels for electron transfer that may have a relation to the channels. Comparing the characteristics of electron tunnels in Fig. 4, several important data are listed as follows. Fig. 4A and Table 1 show that a whole core for iron release is completed by the controlled potentials ranging from q100 to y200 mV, indicating that the ferritin can utilize first electron tunnel with high midpoint-potential Ž E1r2 , y115 mV. on the surface of the shell to pick up the electrons from the electrode for iron reduction. A new experiment in Fig. 4B shows that a whole core can be fully reduced and released by the controlled potentials within 225 mV ranging from y150 to y375 mV again, indicating that the ferritin may utilize a second electron tunnel with midpoint potential Ž E1r2 , y310 mV. to pick up the electrons for complete iron release. A range of relative variable potential in Fig. 4A is greater by 75 mV than that in Fig. 4B, by 150 mV than that in Fig. 4C, and another range in Fig. 4B is greater by 75 mV than that in Fig. 4C, clearly indicating that a factor of about 2 in Fig. 4A,C and about 1.5 in Fig. 4A,B were observed by comparing the range of potentials shifting to negative for a whole iron release. Comparing the curve results in Fig. 4 and Table 1, we conclude that the midpoint potential in Fig. 4B is more negative by 195 mV than that in Fig. 4A, and is more positive by 60 mV than that in Fig. 4C, making sure that AvBFo exists in three-electron tunnels with different midpoint potentials on the surface of protein shell, for picking up electrons for iron reduction from the electrode, and that a range of reduction potential for iron release in the reaction medium or from the electrode controlled by ESD may play a key role in deciding which electron tunnel is utilized for picking up the electrons and transferring them to the core. 3.3. Reaction order and kinetics of iron release In the two experiments in Fig. 5, several factors were considered. Ž1. The controlling potential for iron reduction at y600 mV is lower than that of three-electron tunnel potentials from AvBFo in Fig. 4, so that the electron transfer for iron reduction may be carried out by the threefold channels simultaneously, or the single channel with the lowest potential, as well as the fastest rate of iron release. Ž2. A rate in Fig. 5 is considered as average for complete iron release from the threefold channels simulta-
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Fig. 5. Kinetics and reaction order measurements for complete iron release by the electrode reduction. Curve A: kinetics for complete iron release is defined as zero-order reaction. Rate constant K is 10.5 Fe 3q miny1 AvBFy1 and half-life time t1r 2 is 91.5 min. Curve B: kinetics for complete iron release is defined as zero-order reaction in the presence of extra phosphate. Rate constant K Ž p . is 16.7 Fe 3q miny1 AvBFy1 and half-life time t 1r 2 Ž p . is 54 min. Comparing Fig. 5A,B results, the rate and half-life time for complete iron release of two factors about 1.6 and 0.6 are observed in the presence of and the absence of extra phosphate, respectively, but, both reactions fit zero-order law.
neously. Ž3. The controlled potential is lower than that of several strong reducers to have been reported in kinetic studies in ferritins such as dithionite Žy527 mV., FADH 2 Žy219 mV. Ž33., and H 2 gas Žy421 mV. w34x, so that the electrode is able to supply enough electrons to reduce all iron within the core continuously when the potential of ferritin shifts to negative with the number of iron release increasing. Ž4. Extra phosphate is added into the reaction system for understanding its role in limiting the steps of iron release in the AvBFo . Fig. 5A shows the determination of the kinetics of iron release from a plot of number of iron release per molecular AvBFo vs. time when the reduction potential at y600 mV is controlled by ESD, giving a straight line. A rate of iron release keeps constant as a form of 10.5 Fe 3q miny1 AvBFy1 . Fig. 5B shows a plot of the number of iron release per molecular AvBFo against time, giving a straight line. Average rate is 16.7 Fe 3q miny1 AvBFoy1 . Compared with both rates, we can find that the presence of phosphate accelerates the rate of iron release by a factor of about 1.6. These results are consistent with that of accelerating rate of Fe 2q oxidization within the core by a factor about 2.0 in early core formation in the presence of phosphate w35x.
Table 1 Electrochemical characteristics of electron tunnels on the surface of the shell Curves
Range of controlled potentials for complete iron release ŽmV.
Variable range of the potentials ŽmV.
Midpoint potentials of electron tunnels Ž E1r 2 , mV.
E1r 2 shift to negative ŽmV.
A B C
q100 to y200 y150 to y375 y275 to y425
300 225 150
y115 y310 y370
195 60
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Clearly, the kinetics of complete iron release in Fig. 5 fits a zero-order reaction law. Also, these processes can consider that the rates, and the order for iron release are independent of iron content ranging from 0 to 100% of the original within the core, of the variable structure of phosphate and iron on the surface of the core where it reacts with reducers directly, the width of the channel, and the conformational changes of protein shell, but these depend strongly on the reduction potential of the electron tunnel.
4. Discussion An important step in understanding heme role in the spectral absorbance peaks was recently described by Watt et al. w31,32x. These authors have shown that AvBFo , like HSF and pig spleen ferritin ŽPSF., exhibits featureless absorbance peak in visible spectral range after its heme is removed. In appearance, while AvBFo is reduced Žsee Fig. 3., the heme inside the shell in part come to its surface and contributes to the spectral absorbance dramatic increase because it is located and concentrated on the surface of the shell, where it can directly absorb the light from the spectrophotometer. A rate for increasing heme contents on the surface of the shell corresponds with the conformational changes of the shell when the reduction degree of AvBFo increment and its net charge occurring within the core changes greatly, resulting in the shell making a series of regulation for maintaining complete Fe 2q component to stay within the core, indicating that the conformational changes may not only have an effect on limiting the rate of iron release and keep a stable whole core consisting of Fe 2qrFe 3q component within the core, but also shift the reduction potential to negative, and form a new iron core from apoferritin at a slow rate without phosphate aid. However, so far, the roles of these changes in iron metabolism and storage have been noticed to be unlikely, although a lot of similar results in BF by dithionite reduction were reported w11,18,19x. Recently, a series of reports about redox properties of hemes is described by Niki et al. and Hinnen et al. w27,35–37x. These authors have shown that cytochrome c 3 containing hemes exhibits redox behavior with high electrochemical reversibility at mercury electrode without any mediator aid, and have made sure that the c-type heme groups can accept electrons from the electrode directly, and make up a chain for electron transport among hemes in order w37x. AvBFo contains 12 hemes, with four hemesrelectron channel Žsee Fig. 4., and shows electrode activity Žsee Figs. 2 and 5., indicating that the heme groups may take part in the reversible reaction with the electrode for electron transfer, and the electron tunnel may contain heme component, which is called electron– tunnel–heme ŽETH.. Evidently, it is easily understood that the HSF with no hemes does not give reducing peak at mercury electrode and appears sluggish to the physical
electrode w16x, and the AvBFo with ETHs located on the surface of the shell have capacities to accept electrons directly from platinum electrode and transfer them into the core for iron reduction. Biphasic behavior and two rates for complete iron release have been cited by a pathway of channel diffusion with chemical and biological reducers w11,13,38–40x. As the large size of the reduction electrode, AvBF have to utilize its ETH for accepting electrons for self-iron reduction at constant and slow rate, which is lower than that of dithionite reduction about a factor of 3.6 w38–40x, and fits the zero-order law, clearly indicating that the ETH as a simple mechanism of iron reduction with zero-order reaction may play a key role in limiting the rate of iron release by the electrode reduction, but, the self-regulation of protein shell as a complex pathway with the reducers reduction by concentration diffusion. The presence of phosphate accelerates the rate of Fe 3q release, which shows that it takes part in the reaction of electron transfer with ETH. AvBFo may utilize two pathways of ETH, and the channel for picking up the electrons for iron reduction simultaneously, and one pathway by the channel for iron release in exchange of the other complex. AvBFo has at least three ETHs for iron metabolism and storage Žsee Fig. 4..
Acknowledgements This project was supported by Fujian Provincial Natural Science Foundation of China and by National Educational Committee of China to H.Q.H.
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w25x J. Li, J. Wang, Z. Zhong, Y. Tu, B. Dong, A ferritin of cytochrome b from Azotobacter Õinelandii, Sci. Sinica 10 Ž1979. 1022–1027. w26x B.K. Burgess, D.B. Jacobs, E.I. Stiefel, Large-scale purification of high activity Azotobacter Õinelandii nitrogenase, Biochim. Biophys. Acta 614 Ž1980. 196–209. w27x T. Sagara, S. Nakajima, H. Akutsu, Y. Niki, Heterogenous electron transfer rate measurements of cytochrome c3 at mercury electrodes, J. Electroanal. Chem. 297 Ž1991. 271–282. w28x G.D. Watt, A microcalorimetric procedure for evaluating the kinetic parameters of enzyme-catalyzed reactions: kinetic measurements of the nitrogenase system, Anal. Biochem. 187 Ž1990. 141–146. w29x A.J. Pierik, H. Wassink, H. Haaker, W.R. Hagen, Reod properties and EPR spectroscopy of the P clusters of Azotobacter Õinelandii MoFe protein, Eur. J. Biochem. 212 Ž1993. 51–61. w30x G.D. Watt, An electrochemical methods for measuring redox potentials of low potential proteins by microcoulometry at controlled potentials, Anal. Biochem. 99 Ž1979. 399–407. w31x G.D. Watt, R.B. Frankel, D. Jacobs, H.Q. Huang, G.C. Papaefthymiou, Fe 2q and phosphate interactions in bacterial ferritin from Azotobacter Õinelandii, Biochemistry 31 Ž1992. 5672–5679. w32x G.D. Watt, J.W. McDonald, C.H. Chiu, K.R.N. Reddy, Further characterization of the redox and spectroscopic properties of Azotobacter Õinelandii ferritin, J. Inorg. Biochem. 51 Ž1993. 745–758. w33x H.A. Sober, R.A. Harte ŽEds.., Handbook of Biochemistry 34, Chemical Rubber, Cleveland, OH, 1970. w34x J.M.C.C. Coremans, C.J. van Garderen, S.P.J. Albracht, On the redox equilibrium between H 2 and hydrogenase, Biochim. Biophys. Acta 119 Ž1992. 148–156. w35x Y.G. Cheng, N.D. Chasteen, Role of phosphate in initial iron deposition in apoferritin, Biochemistry 30 Ž1991. 2953–2974. w36x C. Hinnen, R. Parsons, Electrochemical and spectroreflectance studies of the adsorbed horse heart cytochrome c and cytochrome c 3 from D. Õulgaris, Miyazaki strain, at gold electrode, J. Electroanal. Chem. 147 Ž1983. 329–337. w37x J.S. Park, Y. Ohmura, K. Kano, T. Sagara, K. Niki, Y. Kyogoku, H. Akutusu, Regulation of the redox order of four hemes by pH in cytochrome c 3 from D. Õulgaris Miyazaki F, Biochim. Biophys. Acta 1293 Ž1996. 45–54. w38x H.Q. Huang, Z.F. Zhang, L.S. Xue, Comparison of physio-chemical properties of ferritins between pig and horse spleen, Acta Zoologica Sinica 43 Ž1997. 170–177. w39x H.Q. Huang, R.K. Watt, R.B. Frankel, G.D. Watt, Role of phosphate in Fe 2q binding to horse spleen holoferritin, Biochemistry 32 Ž1993. 1681–1687. w40x H.Q. Huang, F.Z. Zhang, Q.M. Lin, R.Y. Zeng, X.H. Qiu, N.H. Huang, L.S. Xu, Z. Ding, Kinetic equations and characteristics of iron release from Azotobacter Õinelandii bacterial ferritin. Acta Biophys. Sinica, in press.
Short communication
Spectroelectrochemical investigation of Azotobacter Õinelandii bacterial ferritin He-Qing Huang
a,)
, Feng-Zhang Zhang a , Liang-Shu Xu a , Qing-Mei Lin b, Jing-Wei Huang c , Ding Zeng a b
a Department of Biology, Xiamen UniÕersity, Xiamen 361005, China Research Center of EnÕironment Science, Xiamen UniÕersity, Xiamen 361005, China c Department of Chemistry, Xiamen UniÕersity, Xiamen 361005, China
Received 3 October 1997; received in revised form 28 October 1997
Abstract Bacterial ferritin of Azotobacter Õinelandii ŽAvBF. contains a function in accepting electrons from platinum electrode directly for complete iron release in the absence of a mediator. The reduction potentials of electron tunnels of y125, y310, and y370 mV for iron release are determined by direct spectroelectrochemical technique, which suggests which should be defined as midpoint potentials of electron–tunnel–heme on the surface of protein shell. A kinetic study for complete iron release by the electrode reduction at y600 mV fits the zero-order reaction law. q 1998 Elsevier Science S.A. Keywords: Azotobacter Õinelandii; Bacterial ferritin; Reduction potential; Electron–tunnel–heme and kinetics of iron release; Spectra and mediatorless
1. Introduction Ferritin is found mostly in animal, plant and bacteria and has clearly been recognized by their physiological functions for iron metabolism and storage w1–4x, amino acid sequence similarity w5–7x, and characteristics of physical chemistry w5,8x. The molecular structure of ferritin consists of protein shell with a highly symmetrical multi˚ in overall diameter w2,5x and of an iron subunits of 120 A core containing a few thousand molecules of hydrated iron phosphate compound w2,9x. In vitro, the kinetics of iron release from ferritin has been studied by the stopped-flow technique w10,11x and the normal spectrophotometry w12– 14x in detail. A mechanism by diffusion manner and channel transport that has been widely accepted by most researchers on ferritin is that the reducers such as sodium dithionite w10,12x or reduced methyl viologen w11x and the chelating agents such as a X – a dipyridyl w10–13x or 1,10phenanthroline w15x must pass through the channel of the shell into the core so that the Fe 3q component of ferritin can be reduced within the core and released out of the shell w10,14x. )
Corresponding author.
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 7 . 0 0 0 9 8 - 6
Horse spleen ferritin ŽHSF. appeared reductive currentless by pulse polarography, and its mineral cores isolated from protein shell showed electrode activity by voltammetry w16x, meaning the shell is sluggish to a physical electrode. On the contrary, AvBF and HSF containing Fe 2q within the core can be directly oxidized by large protein oxidants such as cytochrome c and CuŽII. protein with passing through the channel of ferritin scarcely without any mediator aid, suggesting that there are electron tunnels on the protein shell for transferring electron w17x. Comparing the characteristics of physical chemistry among bacterial ferritin ŽBF., HSF, and plant ferritin w1–4x, BF is known to contain heme Ž6–12 hemerBF. w17,18x showing a , b and soret bands at 557.5, 527, and 425 nm from Azotobacter Õinelandii w5x, at 557, 526, and 417 nm from Rhodobacter capsulatus 37b6 w18x, and at 560, 530, and 417 nm from Escherichia coli K 12, W 2244 w19x and to have higher ratio Ž1:1.2–1.4. structure of phosphate to iron within the core than that Ž1:8–9. of HSF w15,16,20,21x. In addition, oxidized AvBF ŽAvBFo . is a hydrogenase with H 2-uptake activity of 450 H 2rAvBF w21x. The maximum absorbance peaks with an 8-nm shift to red between holoŽ417 nm. and reduced apo-ferritin Ž425 nm. or reduced holo-ferritin Ž425–426 nm. are found w13,19,21x, and their
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spectral properties are tightly related to different procedures of extraction and purification w13x, to variant pH medium w22x, and to Fe 2q numbers within the core w20,22x. Recently, the reduction potentials of y475 mV for heme reduction and of y420 mV for iron core reduction from AvBFo were determined by microcoulometry mediator w23x. A rate of the core potential shifting to negative as a form of y205 mV q Žy115 mVrpH. from HSF is obtained by Watt et al. w23x and Huang et al. w24x. But, the actual role in iron metabolism and storage by the heme, the redox potential, the heme and the electron tunnel still remains obscure. Here, we present evidences revealing that AvBFo contains three midpoint potentials of electron–tunnel–heme ŽETH. having capacities to accept directly electrons from platinum electrode for iron metabolism in the absence of a mediator. The kinetics for complete iron release by the electrode reduction at y600 mV fits the zero-order reaction law. 2. Experimental 2.1. Chemicals and materials The strain OP of A. Õinelandii was a gift from The Center for the Study of Nitrogen Fixation, College of Agricultural and Life Science, University of Wisconsin, Madison, USA. Sodium dithionite and a X – a dipyridyl etc. were from Sigma. Most chemicals for experiments are of analytical or spectral purity. 2.2. Cell culture and AÕBF purification A. Õinelandii was grown in a 100-l fermentor in modified burk medium. The cells were harvested by centrifugation in mid-log phase Ž18 h.. Raw AvBFo was first isolated by the methods of Bulen et al. w4x and Li et al. w25x, and further purified by Burgess et al. w26x as previously described for purifying nitrogenase from A. Õinelandii. The purified AvBF was known to exhibit a single band on the gel electrophoresis ŽPAGE. and show a single peak on the high- performance liquid chromatography ŽHPLC.. 2.3. Protein and metallic element analysis Protein concentration measurement is normally described by the Lowry method. Bovine serum albumin containing 99% purity is used for protein standard. The total number of Fe 3q within the shell is determined by an atomic absorbance spectrophotometer after the ferritin is nitrified, and the Fe 2q numbers within the core is measured by spectrophotometry at 520 nm as previously described by Stiefel and Watt w5x and Watt et al. w23x. 2.4. Apparatus and reduction of AÕBFo An anaerobic electrochemical cell consists of three electrodes and an anaerobic reaction vessel Žsee Fig. 1.. Ž1.
Fig. 1. An anaerobic spectroelectrochemical cell. ŽA. Cell; ŽB. Cuvette; ŽC. Outlet; ŽD. Saturated calomel electrode; ŽE. Isolated platinum counter electrode; ŽF. Working platinum electrode; ŽG. Magnetic stirred bar; ŽH. Magnetic stirrer; ŽI. Inlet; ŽJ. Rubber serum stopper for automatically evacuating and flushing with 99.9% Ar gas. A distance about 0.3–0.4 cm among the electrodes is maintained before use.
Electrodes w1x: a radiometer fiber-tipped saturated ŽKCl. calomel electrode ŽSCE. is used as the reference electrode for the electrochemical synthesis detector ŽESD. w2x. An isolated platinum counter electrode Žanode., w3x a working electrode Žcathode, 20 = 60 mm. of round platinum for supporting high reduction current, and w4x the distance of 0.4–0.5 cm among the electrodes is maintained for obtaining stable reduction current. Ž2. Cell: an anaerobic reaction vessel equipped with water thermostat is designed as a closed reaction cell, which is automatically evacuated and flushed with 99.9% Ar gas using an automatic instrument. UV–visible 240 recording spectrophotometer equipped with an anaerobic cuvette of thermostat filled by the Ar gas is used for recording spectra of AvBF at a controlled potential. The equilibrium position at each experimental point for iron reduction is obtained by the stable residual current, which is monitored by a high differential voltmeter, with the controlled potential for 5–10 min. All reaction system contains 0.01 M Tris–HCl in 0.05 M NaCl, pH 7.0 and mediatorless. All reaction temperature Ž308C. is always maintained by a water thermostat. The mixed solution for iron reduction is stirred by a magnetic stirrer at a constant rate Ž300 rpm.. 2.5. Kinetic studies of complete iron release Three milliliters AvBFo and excess a X – a dipyridyl are mixed by an anaerobic instrument and placed into the cell quickly by perfused gas tight syringe. The controlled potential at y600 mV for iron reduction and release is supplied by ESD. The mixed sample is placed into the anaerobic cuvette filled with 99.9% Ar for total Fe 2q numbers measured by spectrophotometry at 520 nm according to the given reaction time. All controlled potentials are converted into standard hydrogen potential before use. The reaction end is indicated by the maximum number of
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iron release per molecular AvBFo Ž1800 Fe 3qrAvBFo .. According to total iron numbers per molecular AvBFo , a 1:1 ratio of added extra phosphate to iron within the core is prepared to study the effect of phosphate on the rate of complete iron release. 2.6. Midpoint potential measurements of the electron tunnel Four milliliters AvBFo is separated on a G-25 Sephadex column Ž2 = 10 cm. previously equilibrated with 0.025 M Tris–HClr0.15 M NaCl, pH 7.0 for removing free iron and phosphate before use. The AvBFo and excess a X – a dipyridyl are mixed by the instrument for 10 min, then the mixture is placed into the cell quickly under anaerobic condition, and is directly reduced by the controlled potential from ESD. Equilibrium position at each experimental point is monitored by a voltmeter. The reaction end at each curve is completed by the maximum number of Fe 3q release per molecular AvBFo Ž1800 Fe 3qrAvBFo .. The midpoint potential measurement of electron tunnel is calculated from half of maximum numbers of iron release per molecular AvBFo Ž1800 Fe 3qrAvBFo . against the given controlled potential at each new experiment.
Fig. 2. Redox spectra of AvBFo ranging from 380 to 580 nm. Curve A: reduction spectrum of AvBFo with electrode reduction at y600 mV for 2 h. Curve B: reduction spectrum of AvBFo with excess dithionite reduction for 2 h. Curve C: spectrum of AvBFo . Curve D: spectrum of AvBFh o after AvBFrd is at least oxidized by air for 1 h. The AvBFrd is anaerobically separated on the G-25 column for removing unstable Fe 2q and phosphate within the core before being oxidized.
2.7. Spectrum measurement of half-oxidized AÕBF Two milliliters reduced AvBF ŽAvBFr . is oxidized by the air or the mixture gas of 21% O 2 and 79% Ar until the decreasing rate of absorbance keeps constant monitored by a spectrophotometer at 557 nm in 2 min, then, the sample called half-oxidized AvBF ŽAvBFho . is placed into the anaerobic cuvette for spectral measurement ranging from 380 to 580 nm. 2.8. Reduction rate measurement of AÕBFo An 8-ml sample of AvBFo is divided into two Ž4.0 ml each vessel. by a micropipette, then the first sample is placed into an anaerobic cell for reduction with controlled potential at y600 mV. The second one is placed into an anaerobic vessel containing excess dithionite for reduction. The reaction of both samples is carried out at a constant stirred rate Ž300 rpm. and 308C simultaneously, and their absorbances are measured by a spectrophotometer-equipped double light at 557 nm against the indicated time.
3. Results 3.1. Spectra and characteristics of AÕBF Fig. 2C shows clearly that AvBFo exhibits the characteristic absorbance peaks at 557 Ž a ., 526 Ž b ., and 417 ŽSoret band. nm. Fig. 2B,C shows that AvBFr absorbance with dithionite reduction ŽAvBFrd . at spectral range from
380 to 580 nm is higher than that of AvBFo , but, a shift of maximum absorbance peaks to red can be unlikely observed with reducing degree increment due to the complete Fe 2q component still retained within the core. Fig. 2A,B shows that AvBFr by the electrode reduction ŽAvBFre . at y600 mV appears similar the absorbance intensity and curve characteristics to that of AvBFrd . It is well seen that a similar reduction degree and rate of both AvBFrd and AvBFre can be easily obtained and controlled by the chemical reducers and the physical electrode. The plotted results in Fig. 3E,S show the reduction rate of AvBFo as a function of the reaction time monitored at 557 nm. Both projection curves in Fig. 3 indicate that two nearly equal reduction rates for the whole protein shell and the iron core in part by dithionite and the electrode reduction are observed within 40 min, meaning a rate of supplying electrons to the core by dithionite is equal to that the ferritin picks up from the electrode. Fig. 2D shows that the whole absorbance intensity of half-oxidized AvBF ŽAvBFho . at spectral range from 380 to 580 nm is lower than that of AvBFrd or AvBFre ŽFig. 2A,B. and is higher than that of AvBFo ŽFig. 2C., clearly suggesting that AvBFho cannot return reversibly to its original conformation even though the oxidization reaction lasts up to 1 h under the air, because the phosphate and iron component in the part within the core have been lost while the protein is separated in the column before being oxidized. From the curve results analysis in Figs. 1 and 2, we can draw a few interesting conclusions. Ž1. A reduced
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Fig. 3. Reduction rate measurements in the AvBFo monitored at 557 nm. Curve E: electrode reduction at y600 mV for 50 min. Curve S: dithionite reduction for 50 min.
rate of AvBFo is faster than a oxidized that of AvBFrd or AvBFre . Ž2. The oxidization process of AvBFrd and AvBFre exhibits a complex pathway Žsee Fig. 1A–D., which is known to appear at a fast rate for oxidization in initial stage within 40 min, but which cannot reflect a complete process and characteristics of ferritin oxidization because of a series of changes involving the rate of iron release, conformation, and self-regulation of protein shell with the number of iron release increment within the core w21x. Ž3. The AvBFho Žsee Fig. 2D. may be a transitional state between AvBFrd or AvBFre and AvBFo . 3.2. Midpoint potential measurement of electron tunnel The reduced mediator such as reduced methyl viologen or natural red by the concentration diffusion can rapidly reduce the various metallic clusters within the protein w27–29x, but not the physical electrode controlled by the ESD, because most metallic proteins are sluggish to directly pick up electrons from the normal physical electrode w30x. One of the biological functions of the threefold channels from the ferritin is to play an important role in the thoroughfare for exchange of small complex, which has been cited by iron deposition and release between apoand holo-ferritins w2,15,31x, but neither an iron chain nor an iron–phosphate has been reported to be located on the surface of the channel for picking up the electrons from the chemical reducers or the electrode. On the contrary, Fig. 4 shows that the Fe 3q component within the core of AvBFo is directly reduced into Fe 2q composition by the platinum electrode and released out of the shell by a X – a dipyridyl, meaning that there are electron tunnels on the surface of protein shell for picking up the electron from the electrode, and transferring them into the core for iron reduction. A whole iron core with reduction potentials at y420 mV from AvBF w32x and at y215 mV from HSF w24x can be fully reduced by various reducers such as ascorbic acid Žy12 mV., FADH 2 Žy210 mV., FMNH 2 Žy250 mV., reduced methyl viologen
Žy416 mV., and dithionite Žy512 mV. w30,33x. In addition, a factor about tenfold for the rate of iron release is accelerated by a mixed reducers that both reduced methyl viologen and dithionite w11x. As these results above mentioned, an interesting conclusion is drawn that the AvBFo may exist in multi-reduction potentials of the channel and of the electron tunnels on the surface of the shell for iron reduction. The results in Fig. 4A show that the Fe 3q component within the core begin to be reduced into Fe 2q composition while the potential giving q100 mV VS P NHE is obtained by ESD, but the actual, about 5% of original Fe 3q component, is only reduced, and most Fe 3q–phosphate complex with the core is unlikely reduced at the indicated potential. Two additional experimental results have shown that the complex of Fe 2q Žbipy. 3 that have been released out of the protein shell appears reductive currentless by the given potential, and the free complex of Fe 2q ions and phosphate can be unlikely found in the medium when the ferritin is reduced, because none of the reduction current that increases drastically is obtained in the initial stage of iron reduction. In addition, regular kinetic properties for complete iron release have been cited w13x, which these results have clearly indicated, that the reductive current monitored by ESD come from the Fe 3q component reduction within the core, and that a stable remnant current, ranging from 20–100 m A, as well as a constant absorbance monitored at 520 nm by the spectrophotometer within 5 min, shows an equilibrium system of iron reduction for the core reduction at the given potential, and that a more negative potential needs to be obtained by ESD so that the remnant iron within the core can be further reduced as the potential of AvBFo shifts to negative with numbers of released iron within the core increment. AvBFo not only contains the three channels for iron release and
Fig. 4. The midpoint potential measurements of electron tunnels from AvBFo by electrode reduction. The three midpoint potentials of electron tunnel of y115 ŽA., y310 ŽB., and y370 ŽC. mV on the surface of the protein shell are determined by direct spectroelectrochemical technique in the absence of mediator and of other chemical, as well as biological reducers, respectively.
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deposition, but also have the three electron tunnels for electron transfer that may have a relation to the channels. Comparing the characteristics of electron tunnels in Fig. 4, several important data are listed as follows. Fig. 4A and Table 1 show that a whole core for iron release is completed by the controlled potentials ranging from q100 to y200 mV, indicating that the ferritin can utilize first electron tunnel with high midpoint-potential Ž E1r2 , y115 mV. on the surface of the shell to pick up the electrons from the electrode for iron reduction. A new experiment in Fig. 4B shows that a whole core can be fully reduced and released by the controlled potentials within 225 mV ranging from y150 to y375 mV again, indicating that the ferritin may utilize a second electron tunnel with midpoint potential Ž E1r2 , y310 mV. to pick up the electrons for complete iron release. A range of relative variable potential in Fig. 4A is greater by 75 mV than that in Fig. 4B, by 150 mV than that in Fig. 4C, and another range in Fig. 4B is greater by 75 mV than that in Fig. 4C, clearly indicating that a factor of about 2 in Fig. 4A,C and about 1.5 in Fig. 4A,B were observed by comparing the range of potentials shifting to negative for a whole iron release. Comparing the curve results in Fig. 4 and Table 1, we conclude that the midpoint potential in Fig. 4B is more negative by 195 mV than that in Fig. 4A, and is more positive by 60 mV than that in Fig. 4C, making sure that AvBFo exists in three-electron tunnels with different midpoint potentials on the surface of protein shell, for picking up electrons for iron reduction from the electrode, and that a range of reduction potential for iron release in the reaction medium or from the electrode controlled by ESD may play a key role in deciding which electron tunnel is utilized for picking up the electrons and transferring them to the core. 3.3. Reaction order and kinetics of iron release In the two experiments in Fig. 5, several factors were considered. Ž1. The controlling potential for iron reduction at y600 mV is lower than that of three-electron tunnel potentials from AvBFo in Fig. 4, so that the electron transfer for iron reduction may be carried out by the threefold channels simultaneously, or the single channel with the lowest potential, as well as the fastest rate of iron release. Ž2. A rate in Fig. 5 is considered as average for complete iron release from the threefold channels simulta-
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Fig. 5. Kinetics and reaction order measurements for complete iron release by the electrode reduction. Curve A: kinetics for complete iron release is defined as zero-order reaction. Rate constant K is 10.5 Fe 3q miny1 AvBFy1 and half-life time t1r 2 is 91.5 min. Curve B: kinetics for complete iron release is defined as zero-order reaction in the presence of extra phosphate. Rate constant K Ž p . is 16.7 Fe 3q miny1 AvBFy1 and half-life time t 1r 2 Ž p . is 54 min. Comparing Fig. 5A,B results, the rate and half-life time for complete iron release of two factors about 1.6 and 0.6 are observed in the presence of and the absence of extra phosphate, respectively, but, both reactions fit zero-order law.
neously. Ž3. The controlled potential is lower than that of several strong reducers to have been reported in kinetic studies in ferritins such as dithionite Žy527 mV., FADH 2 Žy219 mV. Ž33., and H 2 gas Žy421 mV. w34x, so that the electrode is able to supply enough electrons to reduce all iron within the core continuously when the potential of ferritin shifts to negative with the number of iron release increasing. Ž4. Extra phosphate is added into the reaction system for understanding its role in limiting the steps of iron release in the AvBFo . Fig. 5A shows the determination of the kinetics of iron release from a plot of number of iron release per molecular AvBFo vs. time when the reduction potential at y600 mV is controlled by ESD, giving a straight line. A rate of iron release keeps constant as a form of 10.5 Fe 3q miny1 AvBFy1 . Fig. 5B shows a plot of the number of iron release per molecular AvBFo against time, giving a straight line. Average rate is 16.7 Fe 3q miny1 AvBFoy1 . Compared with both rates, we can find that the presence of phosphate accelerates the rate of iron release by a factor of about 1.6. These results are consistent with that of accelerating rate of Fe 2q oxidization within the core by a factor about 2.0 in early core formation in the presence of phosphate w35x.
Table 1 Electrochemical characteristics of electron tunnels on the surface of the shell Curves
Range of controlled potentials for complete iron release ŽmV.
Variable range of the potentials ŽmV.
Midpoint potentials of electron tunnels Ž E1r 2 , mV.
E1r 2 shift to negative ŽmV.
A B C
q100 to y200 y150 to y375 y275 to y425
300 225 150
y115 y310 y370
195 60
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Clearly, the kinetics of complete iron release in Fig. 5 fits a zero-order reaction law. Also, these processes can consider that the rates, and the order for iron release are independent of iron content ranging from 0 to 100% of the original within the core, of the variable structure of phosphate and iron on the surface of the core where it reacts with reducers directly, the width of the channel, and the conformational changes of protein shell, but these depend strongly on the reduction potential of the electron tunnel.
4. Discussion An important step in understanding heme role in the spectral absorbance peaks was recently described by Watt et al. w31,32x. These authors have shown that AvBFo , like HSF and pig spleen ferritin ŽPSF., exhibits featureless absorbance peak in visible spectral range after its heme is removed. In appearance, while AvBFo is reduced Žsee Fig. 3., the heme inside the shell in part come to its surface and contributes to the spectral absorbance dramatic increase because it is located and concentrated on the surface of the shell, where it can directly absorb the light from the spectrophotometer. A rate for increasing heme contents on the surface of the shell corresponds with the conformational changes of the shell when the reduction degree of AvBFo increment and its net charge occurring within the core changes greatly, resulting in the shell making a series of regulation for maintaining complete Fe 2q component to stay within the core, indicating that the conformational changes may not only have an effect on limiting the rate of iron release and keep a stable whole core consisting of Fe 2qrFe 3q component within the core, but also shift the reduction potential to negative, and form a new iron core from apoferritin at a slow rate without phosphate aid. However, so far, the roles of these changes in iron metabolism and storage have been noticed to be unlikely, although a lot of similar results in BF by dithionite reduction were reported w11,18,19x. Recently, a series of reports about redox properties of hemes is described by Niki et al. and Hinnen et al. w27,35–37x. These authors have shown that cytochrome c 3 containing hemes exhibits redox behavior with high electrochemical reversibility at mercury electrode without any mediator aid, and have made sure that the c-type heme groups can accept electrons from the electrode directly, and make up a chain for electron transport among hemes in order w37x. AvBFo contains 12 hemes, with four hemesrelectron channel Žsee Fig. 4., and shows electrode activity Žsee Figs. 2 and 5., indicating that the heme groups may take part in the reversible reaction with the electrode for electron transfer, and the electron tunnel may contain heme component, which is called electron– tunnel–heme ŽETH.. Evidently, it is easily understood that the HSF with no hemes does not give reducing peak at mercury electrode and appears sluggish to the physical
electrode w16x, and the AvBFo with ETHs located on the surface of the shell have capacities to accept electrons directly from platinum electrode and transfer them into the core for iron reduction. Biphasic behavior and two rates for complete iron release have been cited by a pathway of channel diffusion with chemical and biological reducers w11,13,38–40x. As the large size of the reduction electrode, AvBF have to utilize its ETH for accepting electrons for self-iron reduction at constant and slow rate, which is lower than that of dithionite reduction about a factor of 3.6 w38–40x, and fits the zero-order law, clearly indicating that the ETH as a simple mechanism of iron reduction with zero-order reaction may play a key role in limiting the rate of iron release by the electrode reduction, but, the self-regulation of protein shell as a complex pathway with the reducers reduction by concentration diffusion. The presence of phosphate accelerates the rate of Fe 3q release, which shows that it takes part in the reaction of electron transfer with ETH. AvBFo may utilize two pathways of ETH, and the channel for picking up the electrons for iron reduction simultaneously, and one pathway by the channel for iron release in exchange of the other complex. AvBFo has at least three ETHs for iron metabolism and storage Žsee Fig. 4..
Acknowledgements This project was supported by Fujian Provincial Natural Science Foundation of China and by National Educational Committee of China to H.Q.H.
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