as Quantitative analysis of the frequency spectrum of the radiation emitted by cytochrome oxidase enzymes PHYSICAL REVIEW E, VOLUME 64, 051915 1A. Tuseyaki™™ and JM. Dixon? "Department of Physics, University of Alberta, Edmonton, Alberta, Canada T8G 21 Deparment of Physics, The Unversiy of Warwick, Coventry, CV4 7AL, United Kingdom (Received 19 Decomiber 2000; revised manosenpt received 3 April 2001; published 26 October 2001) [A physical made! ss proposed that provides a quantiaive analysis of the energy emitted by proton flows theoagh mitochondrial walls, The mode! developed is based on biochernical and biophysics! properties of the bedded here groups that are involved inthe electron Fenying mechanism, The estimates ofthe energies at apwoximatsly |] eV and conresponding wavelengths of the near infrared radiation generated, with a peak close 19 900 nm, age extremely well with experimental values, The basi idea in the mecbantem proposed is tha the passage of « proton through the mitochondrial wvall’s gate is linked with the eration ofa virtual proton-eletron par in an excited siate of a hydrogen atom. “The electron is temporarily removed frm the enzyme when the proton arrives atthe gate and is subsequently enzyme eyochcome oxidase and in particular the oposite back atthe enzyime's acceptor si DOE: 10,1103/PhysRevE.64.051915 1. INTRODUCTION All living systems spontaneously emit biophotons [1] in a broad range from thermal radiation in the infrared through visible up o ultraviolet. The intensity of biophotons has been deteoted to range from a few (pliotony’s)icen? up to several hundred (photonsis)em?, In most cases the spectral distribu tion is broad and rather flat except in special situations such as cell division. The oFigin of biophoton zesearch can be traced back to the pioneering work of Gurwitseh [2] and ca be linked to the electromagnetic theory of life proposed by ‘Burr and Northrop [3]. In 1955 Colli er al. [4] succeeding in proving the existence of biophoton emission in the visible region between 390 and 680 nm, Today, bioluminescence is a well documented phenom: enon that can be traced to bacteria whose light emissions can be visible to the naked eye. Various higher organisms such as squid, jelly fish, and fireflies emit light using the enzyme luciferase, Wie note that for fireflies and fishes, this ummines cence serves interorganism communication purposes. Colli's experiments were later supported by the results of Ve- selovskti er al. [5], Ruth and Popp (6), Tarasov et a [7], alt of whom studied different onganisms. Significantly, for the purpose, Viadimiroy and L'vovs [8] detected emissions from isolated’ mitochondria of rat liver that occatted most i tensely whet. conditions were optimum for oxidative phos- phorylation. Zhuravlev er al [9] worked with isolared liver mitochondria, finding that mitochondrial luminescence requires ammonium dihydrogen phosphate (ADP) and oxy- fen, and that uncoupled electron transport (aonphosphorylat- {ng) can contribute to the observed luminescence, Since pho~ tons ate emitted when electrons jump from molecular excited slates to lower energy levels. it should not be too surprising to find that photons are emitted from mitochondria, where “email addtess jtusdéphysalberta.ca 1063-651X/2001/64(5)/051913(7):20.00 64 051915-1 en the prcton leaves the gate PACS number(s): 87.15 Mi, 87.45.Aa, 8715.Ya oxidative phosphorylation and electron transport can provide the energetic pathways to boost electrons into excited stares [10], Interestingly, the last part of Ref. [10] comments on the source of bioluminescence in eukaryotic cells in the follow= ing way: “Unlike bacteria and other organisms displaying macroscopie bioluminescence, however, precise mechanisms for weak eukaryotic photon emissions remain to be discov- cred.” It is our intention in this paper to provide an insight into this question Ina series of studies, spanning a period of some 25 years ‘of research Albrecht-Buehler (AB) demonstrated that living cells possess a spatial orientation mechanism located in the centriole [11-13], This is based on an intricate arrangement of microtubule filaments ia two sets of nine triplets each of which are perpendicular to each other. This arrangement pro- vides the cell with a primitive eye" that allows it to locate the position of other cells within a 2°-3° accuraey in the azimuthal plane and with respect to the axis perpendicular to it. He further showed that electromagnetic signals are the triggers for the cells’ repositioning. It is still largely @ mys- tery how the reception of electromagnetic radiation is ac- complished by the centriole. Another mystery related to these observations is the origia of the electromagnetic radia~ tion emitted by a living cell. Using pulsating infrared signals scattered off plastic beads AB mimicked the effects of the presence of another living cell in the neighborhood. The {question that still remains, which we wish to address in this paper is the source of infrared (IR) radiation speculated by ‘AB to originate in the mitochondria, Mitochondria are the ‘organelles that produce the energy in the form of ATP mol- ecules each of which carries approximately 0.5 eV stored in the high energy phosphate group part of which is due to the electrostatic repulsion between the oxygens in the phosphate group. The synthesis of ATP in the mitochondria is a mul tiple step process relying on the transfer of protons across the mitochondria wall and creating a pt gradient. The explana tion of bow this works brought Mitchell a Nobel Prize in ‘©2001 The American Physical SecielyJ, A, TUSZYNSKI AND J. M. DIXON 26 20 % 10 8001200 1600 ® wavelength om) 040 03s 020. 028+ 020 018: 10. Fraction F 05. 0.003 0 “600729600 800 1000 1700 1200 1300 e ‘Wavelengts () FIG. 1. {a} The percentage of colls that removed the light seat reting particle as a function of wavelength. The most “attractive” wavelength wis between 800 and 900 nm, (hitp? wivw:basic.awedu’g-buehlevirvision.hr}. (b) The fraction F of cells that remove the light seattring particles. 1971 [14], At the center of this mechanism isthe functioning, ‘of proton pumps embedded in the mitochondrial al Broadly speaking, these pumps are intricately built with aa active moveable part consisting of an enzyme called cyto chrome oxidase that opens and closes u channel through which individual H ions enter the mitochondria, & living cell is @ nonegilibrinm physical system that requires @ con: tinuous energy input to sustuin its activities. In animal cells energy production invalves breakdown of nutrients and a subsequent production of ATP molecules that are the univers sal currency of energy in cells, The production of ATP mol ‘ecules in tum is directly linked to the regular functioning of the mitochondrial pumps. It is therefore logical that one of the signatures of a living cell would be related to the osci latory (pulsating) effect of mitochondrial proton flow. AB did suggest that the mitochondria are the best candidates for the explanation of infrared emission effects, He even pointed in the direction of the porphyrin containing proteins, .e.. the cytochromes, It is our intention in this paper w provide a guantilative account of how the structure of the porphyrin tnolecule results in the spectrum of emitted IR radiation ub- served in AB's experiments, Based on statistical analysis of culture of 800 cells AB found a response function to the wavelength of electromaynetic radiation found below. The peak in Fig, | appears to coincide with 850 1m hac is in the rear IR and in the following pages we propese « detailed {quantitative mechanism explaining suck an emission spec~ 45191 PHYSICAL REVIEW E 64 051915 Membrane FIG, 2. A mae of the transducing components located on the inner side ofthe mitochondrial membrane The effects of biophioton emission and absorption in liv ing cells cover a very large body of literature, as we have shown above, some of which provides corroborative, albeit indirect, evidence for studies of ATbrecht Buehler. II, THE BIOCHEMICAL STRUCTURE AND FUNCTION OF THE CYTOCHROMES ‘The mechanisms involved in the process of proton gradi- cents are illustrated in Fig, 2. To convert the energy of redox reactions of photoredox processes into a movement of pro- tons from the leit- to the right-hand side of the membrane, the redox driven protonmotive system is postulated. The separation of protons and anions across the membrane holds the energy and the stored potential energy is utilized in (wo ‘ways When there ‘s a right-to-left flow of protons through the ATP, synthetase is coupled to the eneriy requiring syn- thesis of ATP from ADP and inorganic phosphate. This later tow is coupled to wctive transport of biochemical compo nents, The reverse flow involves the hydrolysis of ATP on. the left side accompanied by the left-to-right proton flow ATP synthetases are located asymmetrically in_mem- branes that are driven by the electrochemical potential of the protons and have a HW/P stoichiometry characteriving it ‘The major subunits are shown in Fig. 3. To the left ‘eytuchrome-c-oxidase and to the right the quinol oxidase. In both cases the motif of a single heme plus heme/copper bi- FIG. 3. A schematic diagram of eytachrome c oxidase (eyt.a83)QUANTITATIVE ANALYS! I$ OF THE FREQUENCY nuclear center is evident. An analogy may be drawn, despite the fact that the enzymes differ in the neture oftheir electron entry sites, hetwween electron donation from cytochrome ¢ t0 the Cu, /eytochrame « sites and from quinal (QH) to heme. The oxidation system in mitochondia is made up, prine: pully, of the membrane associated with the electron transport chain ‘The oxidation of reduced NAD, the reduction of O2 10 ‘water that accompanies it, and the storage of energy ane the overall function of this system, It is assumed in the chemi- fosinotic hypothesis that the intermediate form is created by transporting hydrogen ions from the inner mitochondrial ma~ trix to the exterior of the inner mitochondrial membrane. A representation of the process in the mitochondria can be schematically illustrated by the following reactions: L NADH+H"~ 5 0s'SNAD*+H.O+ESE, (2.1) ADP+ P+ ESE=ATP. (2.2) ‘The electrochemically stored energy is represented by SB. The reaction in Eq, (2.1) deseribes the oxidation sub- system and ESE is associated with the membrane. The pro- cess in Eg, (2.2) deseribes the transduction system. Available models have restrictions placed upon them by experimental features. The reactions ip Eqs. (2.1) and (2.2) can be thought of as reversible since, in the presence of excess ATP and an appropriate electron source other than water, the reactions ‘can be run backwards Ieading (@ such high energy reduced intermediates as NADH. This mechanism seems to be pos- sible in many systems and a normal pathway in others. A. second reason for reversibility is that respiratory control of the rate of oxygen utilization by ADP suggests a near-to- equilibrium distribution of the products of the first reaction By a utilization of ESE the reaction is shifted so the right. Clearly the reversible and neat-to-cquilibrium features sug- ‘gest that equilibrium theemodynamics can be used, We may assuine that for Eq, (2.2) the actual sites for the correspond ing processes are the ATP synthetase complexes of the inner ‘mitochondrial membrane, ‘As a super family of metalloenzymes, the termival oxi ddases share the basie function of catalyzing, by a four elee- tron process, the complete reduction of oxygen to water. For our purposes itis important to emphasize that a proton elec: trochemical gradient is established across the membrane by the terminal oxidases. This is achieved by taking up the re- quired protons to produce water from one side of the mem- brane only, These latter oxidases have ulso been shown to amp protons across the membrane. This occurs in a provess that implies « coupling, against the diffusion grudient, be- tween endergonie proton translocation and an exergonie tlectton transfer process. The terminal oxidases, therefore, contribute significantly to the maintenance of a transmem- brane proton electrochemical gradient. Crystallographic structures of cytochrome oxidase enzymes have been cstab- lished and we show one example below in Fig. 4 Tr has been firmly established that there 1s a newr-zero ‘activation energy in oxidation of reduced cytochrome ¢ by PHYSICAL REVIEW F 64 051915 FIG, 4, A view from the mitochondria intermembrane space down the membrane normal: a stsrsoview of eytocirome ¢, subunit 8, and the metaleluster binding fold of the ISP atthe “c,"" state. ‘The disulphide bonds in subunit 8 are shown and ligands of heme His of ISP ferricyanide, The uptake of an electron at the iron center or the energy for release may come from the vibrational energy of the protein, Furthermore for our purposes in this paper, it would seem quite probable that conformational mobility of the protein may assist in the electron-transter process and the control of the gating process itself. The removal of sites for binding ferricyanide and elimination of surface positive charges via modification of lysine residues, does not affect, the activation energy that remains near zero. The treatment of the protein with 4M guanidine hydrochloride (which re laxes the binding of ferracyanide 1 the reduced molecule) does not aflect the electron transfer rate. The notion that conformational changes are important in the electron transfer function of cytochrome ¢ is supported by the above ev dence. ‘At the center of the enzyme one always finds am all im- portant heme group that plays a cardinal functional role. The hheme group, which is essentially a network of 36 conjugated bonds arranged in @ flat disc, is derived ftom porphyrin mol ecules. Below we show the basic structure of the heme group in Fig, S(a) as well as specific example in Fig. 5(6). Iwata eral [15] pointed out that the structure of the cytochrome ‘be, complex, especially the iton-sulphur complex suggests the existence of a new electron transport mechanism of the enzyme. The importance of electroconformational coupling, jie. the influence of oscillating electric fields for the en- zymes cellular enerey production lias been demonstrated by Tsong er al [16] within a physical framework. The heme ‘group and telated molecules often contain a metal ion at their center. One can imagine that such systems of conjugated bonds will exhibit physical properties that are somewhere between" those produced by the energy levels of single jons and the continuous energy band structure of solid state 0519153