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 H2O2.
CII formation is directly related to UVB intensity and CAT concentration, proceeding at a constant rate of 0.024 CII nmol/CATheme nmol/ml/min under UVB intensity of 450 µW/cm2. As a comparison, CII generates at rate of 0.021 nmol/CAThemenmol/ml/min in the presence of NS producing 2 H2O2 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 H2O2 production follow the same kinetics (Fig. 1c), which supports that H2O2 photoproduction is inhibited by inactive CII formation. A conformational mechanism could contribute to the link of H2O2 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 H2O2 break down inhibits H2O2 photoproduction, which given the harmful nature of H2O2 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 H2O2 levels that occur naturally in cells. NADPH is oxidized in this process at rates that depend on the rate of H2O2 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/CAThem nmol/min under UVB intensity of 450µW/cm2. NADPH also decreases CII concentration at plateau in a concentration-dependent manner, which results in a similar decrease in H2O2 levels (Fig. 1d). Thus, NADPH inhibiting CII formation achieves a new steady state in which CATUVB produces and decomposes more H2O2 (Fig. 1d) in a way that CATUVB activity would naturally be driven by NADPH concentrations.
Now the question is: why does CATUVB generate H2O2 only then to break it down? In other words: what is CAT actually doing in cells under solar UVB? CATUVB uses O2 and H2O to produce H2O2 (Heck et al. 2003), which is then decomposed into H2O and O2. That is, CATUVB performs a cyclic reaction that does not produce any chemical energy, so given that light emission (i.e. fluorescence) by CAT excited by UVB is negligible (Yekta et al. 2017), all UVB used for H2O2 synthesis finally becomes heat. Assuming that one H2O2 molecule is produced per UVB photon absorbed, the energy of one einstein of UVB photons at 300 nm (Emol = Nhν) being around 400 kJ would be the heat released per mol of H2O2 generated and decomposed by CATUVB.
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 significant 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 first 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 specific 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 cm2, 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/cm2. 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; Kasting and Siefert 2002; Sinha and Häder 2002; McKencie et al. 2002; Karam 2003; Hessen 2008). Data have shown here that CAT converts UVB into heat at very low temperatures (Fig. 2), which reveals the role of CATUVB in nature. That is, CATUVB producing beneficial 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 eons, 3,5 Gy ago (Zámocký et al. 2012), precisely when microorganisms were subjected to high-intensity UVB due to the low levels of atmospheric O2 (McKencie et al. 2002; Karam 2003) and an extremely cold climate (Kasting and Siefert 2002), including several low-latitude glaciations episodes known as Snowball Earth (Hoffman et al. 1998; Kirschvink et al. 2000;). Furthermore, evidence for H2O2 photoproduction during such cold periods supports CATUVB activity, whose thermogenesis might pave the way to the rise of oxygenic photosythesis (McKay and Hartman 1991; Kasting and Siefert 2002; Liang et al. 2006). In turn, oxygenic photosynthesis being the main NADPH and O2/O3 producer would become the primary driver of CATUVB thermogenesis (Fig. 4).
B) CATUVB playing a biogeochemical process with impact on ice melting and sea warming
Thermogenesis due to an unknown UVB-driven H2O2 synthesis and its decomposition by CAT in microorganisms has been previously proposed as a photocalytic factor in sea ice melting and sea warming in cold regions with severe ozone depletion (Moreno 2012). Experimental evidence for that UVB thermogenesis (Fig. 2) in microorganisms is now evidence for such a hypothesis. Vice versa, CATUVB activity in the enormous mass of microbes living in sea ice (SIMCO) (Palmisano and Sullivan 1983; Smith and Nelson 1986; Kottmeier and Sullivan 1988), sea ice edge (Smith and Nelson1986; Russell 1990) and sea surface of cold regions (Behrenfeld et al. 2006; Sigman and Hain 2012) would be responsible for a process of biogeochemical thermogenesis under the ozone layer control with effects on ice melting and sea warming in cold regions.