Catalase Catalyses the Conversion of Ultraviolet-B Radiation Into Heat: Unexpected Role for Microorganisms in Sea Ice Melting and Sea Surface Warming

Catalase under ultraviolet-B radiation (CATUVB) produces reactive oxygen species (ROS), but the role of that surprising photoactivity in CAT still remains uncertain. On the other hand, it is well-known that CAT breaking down a steady source of hydrogen peroxide (H 2 O 2 ) becomes inactive due to compound II formation (CII), which results in typical changes in its absorption spectrum. On the basis of CII formation, here I show rst that CATUVB produces and breaks down H 2 O 2 , via which UVB is converted into heat. I then show that CATUVB thermogenesis accelerates the ice melting and warms the medium. From data to nature, CAT converting harmful UVB into advantageous heat in microorganisms would be responsible for a hidden biogeochemical thermogenesis process under the ozone layer control with effects on sea ice melting and sea surface warming in cold regions.


Introduction
It is widely accepted that CAT breaks down H 2 O 2 from cellular metabolism, but some believe the ultimate role of such a venerable enzyme may still be a mystery (Kirkman and Gaetani 2007). In support of this view, it was unexpectedly discovered that CAT is a photoactive enzyme, responsible for the production of ROS in cells exposed to UVB (Heck et al. 2003). UVB-driven photoactivity has so far been shown in CAT with bound NADPH (Kirkman and Gaetani 1984;Fita and Rossmann 1985) from mammals and bacteria (Heck et al. 2010). Regarding the ROS product, preliminary data excluding superoxide anion and singlet oxygen rather supported "peroxide" species, likely H 2 O 2 (Heck et al. 2003(Heck et al. , 2010. Whether or not CATUVB produces H 2 O 2 remains unresolved, probably because H 2 O 2 is the substrate of CAT, one of the fastestacting enzymes in biology. So, with the lack of further data, the role of CATUVB still remains uncertain. On the other hand, it has long been known that CAT exposed to a constant H 2 O 2 source, as generated by the notatin system (NS) (i.e. glucose oxidase/glucose), becomes progressively inactivated due to CII formation (Chance 1950; Keilin and Nicholls 1958;Kirkman et al. 1987). CII formation during the CAT catalytic cycle can be summarized as follows: the resting CAT reacts with H 2 O 2 yielding H 2 O and compound I (CI), whose reaction with a second H 2 O 2 yields O 2 , H 2 O and the resting enzyme. However, when decomposing a steady source of H 2 O 2 , CI is progressively one electron oxidized by an endogenous electron donor generating inactive CII, which causes a unique increase in absorption at 435 nm. Typically, CII formation is strongly accelerated by an exogenous electron donor, such as ferrocyanide, and reverses spontaneously (Chance 1950;Kirkman et al. 1987). Thus, if CII formation invariably occurs when CAT is exposed to a source of H 2 O 2 , CII formation in CATUVB would be evidence for H 2 O 2 photoproduction, which could pave the way to a better understanding of the role of CATUVB.
A UVB transilluminator CU10A (CLINX) with a maximal intensity of 800 µW/cm 2 at 302 nm was used for irradiation. UVB intensity was measured by a radiometer UV340B (Sampometer). Temperature was measured by a Center 306 dual datalogger thermometer (Center Technology Corp.) with a resolution of 0.1 ºC, using K type beaded thermocouple probes (TP-K01). A UV-VIS 1200 spectrophotometer was used for all purposes.

CAT preparation
Crystallised CAT suspension in 0.1 % thymol was directly dissolved in 20 mM potassium phosphate buffer, pH 7.4, for 30' and centrifuged 10' at 10.000 rpm to remove any precipitate (Hillar and Nicholls 1992). CAT concentration was measured in terms of haematin content, considering an extinction coe cient of 120 mM − 1 cm − 1 at 405 nm (Hillar et al. 1994).

Irradiation procedure
Samples, usually of 1ml, inside quartz or polymethylmethacrylate (PMMA) cuvettes were irradiated in horizontal position from above. Only one light bar of the transilluminator was used, so cuvette and bar were aligned along their major axis. The desired UVB intensity was achieved with the transilluminator potentiometer or by placing the samples at a suitable distance from the transilluminator surface.
CII formation and NADPH oxidation measurement CII formation by UVB irradiation was measured by the increase in absorbance at 435 nm, as previously described (Chance 1950, Kirkman et al. 1984). Kinetics of CII formation versus irradiation was carried out by irradiating one CAT sample and measuring the absorbance at given times. CII concentration was calculated considering an extinction coe cient of 32 mM − 1 cm − 1 (Chance 1950, Kirkman et al. 1984.

Observation of CATUVB thermogenesis
For the temperature-controlled UVB irradiation experiments, silicone-sealed PMMA cuvettes containing CAT and control samples were housed in cavities made of expanded polystyrene (EPS) modules, so that only the surface to be irradiated was free of insulation. Special care was taken so that temperature changes were identical in both cuvettes in any condition, in particular under UVB irradiation given that the UVB-transilluminator is a signi cant heat source. Temperature was measured by a K type beaded thermocouple probe (TP-K01) with te on tape insulation, inserted into the cuvette through the silicone stopper. For experiments at temperatures below 0º C, CAT and control samples were rst frozen at -19º C and then allowed to melt spontaneously at room temperature of 13 ºC, with UVB irradiation starting at -5ºC. For experiments at temperatures up 0º C, CAT and control samples at room temperature of 13º C were irradiated until the temperature increased to 5º C (i.e. above 18º C).

Data processing
Data were processed with Graph Pad Prism. Unless stated differently, measurements were carried out in duplicate (mean ± SD). Temperature charts were directly obtained by unloading logged temperature data into a PC interface with Windows software (SE 305, version 4.8.0.0). Schematic gures were carried out with Power Point from Microsoft.

Results And Discussion
CII formation by CATUVB CATUVB experiences an immediate increase in absorption at 435 nm (Fig. 1aA). More importantly, this increase in absorption is strongly accelerated by ferrocyanide (Fig. 1aB). Moreover, consistent with CII formation, the increase in absorption is reversible upon cessation of UVB irradiation (Fig. 1a). UVB-driven CII formation is also evident in full CAT spectra (Chance 1950, Kirkman et al. 1987, especially in the presence of ferrocyanide, where most of the CAT is converted to CII (Fig. 1b). Consequently, UVB-driven CII formation shows that CATUVB produces and breaks down H 2 O 2 .
CATUVB kinetics CII formation is directly related to UVB intensity and CAT concentration, proceeding at a constant rate of 0.024 CII nmol/CAT heme nmol/ml/min under UVB intensity of 450 µW/cm 2 . As a comparison, CII generates at rate of 0.021 nmol/CAT heme nmol/ml/min in the presence of NS producing 2 H 2 O 2 nmol/ml/min (Kirkman et al. 1987). CII formation is initially linear, decreasing slowly to reach a plateau after 40' of UVB irradiation (Fig. 1 aA). Since CII decays spontaneously (Chance 1950;Kirkman et al. 1987), such a plateau represents a steady state where CII formation and decomposition occur at the same rate. Of note, CII formation and H 2 O 2 production follow the same kinetics (Fig. 1c), which supports that H 2 O 2 photoproduction is inhibited by inactive CII formation. A conformational mechanism could contribute to the link of H 2 O 2 photoproduction and CII formation, as the latter's spectral change (Fig. 1b) must entail a conformational change in CAT. In summary, inactive CII formation due to H 2 O 2 break down inhibits H 2 O 2 photoproduction, which given the harmful nature of H 2 O 2 would be essential to prevent CATUVB toxicity in cells.
The effect of NADPH on CATUVB It has been exhaustively shown that NADPH (but not NADH) traces inhibit CII formation or accelerate its decay in CAT exposed to NS (Kirkman et al. 1987;Hillar and Nicholls 1992;Hillar et al. 1994), so it is believed that NADPH plays a physiological role in keeping CAT catalatic activity at H 2 O 2 levels that occur naturally in cells. NADPH is oxidized in this process at rates that depend on the rate of H 2 O 2 production by NS, both processes being not at all stoichiometric (Kirkman et al. 1987). NADPH is also oxidised by CATUVB at a constant rate of 0.13 NADPH nmol/CAT hem nmol/min under UVB intensity of 450µW/cm 2 .
NADPH also decreases CII concentration at plateau in a concentration-dependent manner, which results in a similar decrease in H 2 O 2 levels (Fig. 1d). Thus, NADPH inhibiting CII formation achieves a new steady state in which CATUVB produces and decomposes more H 2 O 2 (Fig. 1d)  The effect of CATUVB on the medium temperature CATUVB releasing about 13 times the free energy of ATP hydrolysis (i.e. 30,5 kJ/mol) would be by far the most exothermic process in biology. Warm temperature is essential for sustaining life processes, so that powerful CATUVB thermogenesis would only make sense if CATUVB works in a cold environment. In fact, ROS output by CATUVB was observed to proceed at signi cant rate at 0 ºC (Heck 2003). Accordingly, CATUVB could produce heat from UVB even at very low temperatures, which could be experimentally observed. Thus, in experimental conditions where CAT and control samples were rst frozen between − 19º and − 15º C and then allowed to warm at room temperature of around 13ºC, with UVB irradiation starting at -5ºC (see Methods), data show a difference in temperature in CAT samples with regard to controls peaking at between − 1,0 and 1,0 ºC (Fig. 2a). So, CATUVB thermogenesis would be contributing to the speci c heat of ice melting. Likewise, when irradiated at 13ºC, the increase in temperature is faster in CAT samples than in controls, with a difference of around 0,2 − 0,3 ºC at 20 minutes (Fig. 3b). Given the experimental conditions involving an irradiation surface of 1,2 cm 2 , 0,8 ml of sample volume and CAT concentration enough as to absorb most UVB radiation at 300 nm, about 0.8 J would be needed for a such difference in temperature, which is consistent with the 0.86 J delivered in 20 minutes by the UVB-transilluminator at an intensity of 600 µW/cm 2 . Ferrocyanide made possible the experimental observation of CATUVB thermogenesis, due to CATUVB producing more heat in its presence than alone or in the presence of NADPH.
The role of CATUVB: from data to nature A) CATUVB and life evolution It is well-known that UVB is harmful to cells and that most of the catalytic reactions that sustain life are cold-sensitive. However, life on Earth has evolved under extremely adverse cold and UVB conditions, most of the time acting together (Russell 1990 (Fig. 2), which reveals the role of CATUVB in nature. That is, CATUVB producing bene cial heat from harmful UVB, is responsible for an unprecedented thermogenesis that allows cells to evolve under adverse cold and UVB conditions (Fig. 3). In this regard, CAT in known to evolve in Precambrian

Conclusions
Experimental evidence based on CII formation has shown that CATUVB produces and breaks down H 2 O 2 , a highly regulated photocatalysis under control of NADPH whose ultimate effect is the transformation of UVB into heat. Moreover, it is shown here that such UVB-driven thermogenesis accelerates ice melting and warms the medium. CATUVB would allow microorganisms to evolve in adverse cold and UVB conditions. Furthermore, that UVB-driven thermogenesis in the huge mass of microbes living in sea ice and sea surface would be responsible for a new ozone layer-driven biogeochemical process with effects on the biosphere evolution. Figure 1 a) Absorption increase at 435 nm in CAT (4 µM CATheme under UVB intensity of 450 mW/cm2) showing CII formation. A: CAT alone. B: in the presence of 50 µM ferrocyanide. One sample was used for each curve. Arrow heads indicate end of UVB irradiation. b) CAT spectrum (5,7 µM CATheme) from 380 to 460 nm and its spectral changes after 40 minutes of UVB irradiation, and in the presence of ferrocyanide (50µM) after 5 minutes. Arrows point to the absorbance increase at 435 nm (vertical line) showing CII formation. c) ROS/H2O2 production by CATUVB (1µM CATheme). Absorbance at 504 nm was measured 24 hours after irradiation. d) The effect of NADPH on CII formation by CATUVB (4 µM CATheme under UVB intensity of 450 mW/cm2). CII formation at plateau was measured in CAT samples containing different NADPH concentrations. Inset: The effect of NADPH on ROS/H2O2 production by CATUVB (1µM CATheme). Same procedure as for CII formation. Absorbance at 504 nm was measured 24 hours after irradiation.

Figure 2
Experimental observation of the transformation UVB into heat by CAT (20 µM CAThem in 20 mM phosphate buffer pH 7,4, 50 µM ferrocyanide. Samples of 0,8 ml in silicon sealed PMMA cells of 1x1x1,2mm under UVB intensity 600 µW/cm2. a) Effect of CATUVB on ice melting. Samples were frozen at -19ºC and then allowed to melt at 13ºC. UVB irradiation started at -5ºC. Black line: Temperature recording of CAT sample. Red broken line: temperature recording of control sample. Inset: Temperature recording of two control samples following the same procedure. b) Effect of CATUVB on the temperature of the medium. Samples equilibrated at 13,6 ºC were subjected to UVB irradiation until the temperature increased to 19ºC. Black and red lines as aforementioned. Inset: Temperature recording of two control samples following the same procedure.