The Photoactive Excited State of the B₁₂-Based Photoreceptor CarH

Abstract

We have used transient absorption spectroscopy in the UV–visible and X-ray regions to characterize the excited state of CarH, a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light. With visible excitation, a nanosecond-lifetime photoactive excited state is formed with unit quantum yield. The time-resolved X-ray absorption near edge structure difference spectrum of this state demonstrates that the excited state of AdoCbl in CarH undergoes only modest structural expansion around the central cobalt, a behavior similar to that observed for methylcobalamin rather than for AdoCbl free in solution. We propose a new mechanism for CarH photoreactivity involving formation of a triplet excited state. This allows the sensor to operate with high quantum efficiency and without formation of potentially dangerous side products. By stabilizing the excited electronic state, CarH controls reactivity of AdoCbl and enables slow reactions that yield nonreactive products and bypass bond homolysis and reactive radical species formation.

Controlling Non-Native B12 Reactivity and Catalysis in the Transcription Factor CarH

“…CarH is B12-dependent transcription factor that exploits a remarkable exaptation of AdoCbl Co(III)-carbon bond cleavage 18,19 to enable light-activated gene expression, 15 rather than the more common radical rearrangement processes catalyzed by other AdoCbl-dependent enzymes (Figure 1B) 2 . In its AdoCbl-bound form, CarH forms a tetramer that binds DNA to repress transcription of carotenoid biosynthesis genes that protect organisms from light damage.…”

Light-Triggered Carotenogenesis in Myxococcus xanthus: New Paradigms in Photosensory Signaling, Transduction and Gene Regulation

“…Both homologs turned out to be B 12 -dependent like CarH. Studies of the homolog in the Gram-negative Thermus thermophilus , CarH Tt , yielded valuable biochemical [ 20 ], structural [ 19 ] and photochemical insights [ 61 , 62 , 63 ] that were further extended with CarH Bm , the homolog in the Gram-positive Bacillus megaterium [ 64 , 65 ]. These findings, reviewed elsewhere [ 21 , 22 ], are briefly highlighted here.…”

“…Its enzyme-catalyzed cleavage, which enables the use of AdoCbl in mutases, dehydratases, deaminases and ribonucleotide reductases and of MeCbl in methyltransferases, has been extensively studied and reviewed elsewhere [ 54 , 55 ]. Cleavage of the Co-C bond, by near-UV and visible light of wavelengths <530 nm, also underlies the use of AdoCbl as a chromophore for light sensing and response by CarH proteins ( Figure 2 b,c), which now represent a separate, large and widespread photoreceptor family among the ten currently known [ 19 , 20 , 21 , 22 , 61 , 62 , 63 ].…”

“…The photochemistry of CarH Tt -bound AdoCbl examined by analyzing photolysis products [ 61 ], by ultrafast spectroscopy [ 62 , 63 ] and by theoretical calculations [ 67 ] suggested it may differ significantly from that established for free or enzyme-bound AdoCbl. Photolytic cleavage of free AdoCbl, often a model for that in AdoCbl-dependent enzymes, is homolytic and generates reactive cob(II)alamin and Ado• radical species that rapidly react to yield specific products depending on the presence or otherwise of molecular oxygen [ 21 , 61 , 68 ].…”

“…It was found that photolysis of CarH Tt -bound AdoCbl avoids release of Ado• radicals by generating 4′,5′-anhydroadenosine, a harmless product undetected upon cleavage of free or enzyme-bound AdoCbl ([ 61 ]; Figure 2 c). Based on ultrafast spectroscopy data, it has been proposed that CarH enables an unprecedented heterolytic cleavage of the AdoCbl Co–C bond to bypass radical formation and release [ 62 ] or stabilizes an excited state long enough to ensure the reactions that yield the 4′,5′-anhydroadenosine product [ 63 ]. The molecular mechanism for how CarH alters AdoCbl photochemistry is still unclear.…”