Flavodoxin hydroquinone reduces Azotobacter vinelandii Fe protein to the all-ferrous redox state with a S = 0 spin state

Abstract

Azotobacter vinelandii flavodoxin hydroquinone (FldHQ) is a physiological reductant to nitrogenase supporting catalysis that is twice as energy efficient (ATP/2e^– = 2) as dithionite (ATP/2e^– = 4). This catalytic efficiency results from reduction of Fe protein from A. vinelandii (Av2) to the all-ferrous oxidation state ([Fe₄S₄]⁰), in contrast to dithionite, which only reduces Av2 to the [Fe₄S₄]¹⁺ state. Like FldHQ, Ti(III) citrate yields ATP/2e^– = 2, and Ti(III)-reduced [Fe₄S₄]⁰ Av2 has a S = 4 spin state and characteristic Mossbauer spectrum, a parallel mode g = 16.4 EPR signal, and a shoulder at 520 nm in its UV-vis spectrum, each of which distinguish the S = 4 [Fe₄S₄]⁰ Av2 from other states. In this study, we demonstrate that FldHQ makes [Fe₄S₄]⁰ Av2, which is sufficiently characterized to demonstrate unique physical properties that distinguish it from the previously characterized Ti(III)-reduced [Fe₄S₄]⁰ Av2. In particular, Evans NMR magnetic susceptibility and EPR measurements indicate that FldHQ-reduced [Fe₄S₄]⁰ Av2 has an S = 0 spin state (like [Fe₄S₄]²⁺ Av2). There is no g = 16.4 EPR signal and no shoulder at 520 nm in its absorbance spectrum, which resembles that of [Fe₄S₄]¹⁺ Av2. That the physiological reductant to Av2 is capable of forming [Fe₄S₄]⁰ Av2 has important implications for in vivo nitrogenase activity.